Promoter, promoter control elements, and combinations, and use thereof

ABSTRACT

The present invention is directed to promoter sequences and promoter control elements, polynucleotide constructs comprising the promoters and control elements, and methods of identifying the promoters, control elements, or fragments thereof. The invention further relates to the use of the present promoters or promoter control elements to modulate transcript levels in plants, and plants containing such promoters or promoter control elements.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to prior U.S. provisionalapplication Ser. No. 61/025,697, filed on Feb. 1, 2008, the entirecontents of which are hereby incorporated by reference.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING OR TABLE

The material in the accompanying sequence listing is hereby incorporatedby reference into this application. The accompanying file, namedSequence_Listing_(—)2750-1720WO1.txt was created on Dec. 22, 2008 and is56 KB. The file can be accessed using Microsoft Word on a computer thatuses Windows OS.

FIELD OF THE INVENTION

The present invention relates to promoters and promoter control elementsthat are useful for modulating transcription of a desiredpolynucleotide. Such promoters and promoter control elements can beincluded in polynucleotide constructs, expression cassettes, vectors, orinserted into the chromosome or as an exogenous element, to modulate invivo and in vitro transcription of a polynucleotide. Host cells,including plant cells, and organisms, such as regenerated plantstherefrom, with desired traits or characteristics using polynucleotidescomprising the promoters and promoter control elements of the presentinvention are also a part of the invention.

BACKGROUND OF THE INVENTION

This invention relates to promoter sequences and promoter controlelement sequences which are useful for the transcription ofpolynucleotides in a host cell or transformed host organism.

The introduction of genes into plants has resulted in the development ofplants having new and useful phenotypes such as pathogen resistance,higher levels of healthier types of oils, novel production of healthfulcomponents such as beta-carotene synthesis in rice. An introduced geneis generally a chimeric gene composed of the coding region that confersthe desired trait and regulatory sequences. One regulatory sequence isthe promoter, which is located 5′ to the coding region. This sequence isinvolved in regulating the pattern of expression of a coding region 3′thereof. The promoter sequence binds RNA polymerase complex as well asone or more transcription factors that are involved in producing the RNAtranscript of the coding region.

The promoter region of a gene used in plant transformation is most oftenderived from a different source than is the coding region. It may befrom a different gene of the same species of plant, from a differentspecies of plant, from a plant virus, an algae species, a fungalspecies, or it may be a composite of different natural and/or syntheticsequences. Properties of the promoter sequence generally determine thepattern of expression for the coding region that is operably linked tothe promoter. Promoters with different characteristics of expressionhave been described. The promoter may confer broad expression as in thecase of the widely-used cauliflower mosaic virus (CaMV) 35S promoter.The promoter may confer tissue-specific expression as in the case of theseed-specific phaseolin promoter. The promoter may confer a pattern fordevelopmental changes in expression. The promoter may be induced by anapplied chemical compound, or by an environmental condition applied tothe plant.

The promoter that is used to regulate a particular coding region isdetermined by the desired expression pattern for that coding region,which itself is determined by the desired resulting phenotype in theplant. For example, herbicide resistance is desired throughout the plantso the 35S promoter is appropriate for expression of anherbicide-resistance gene. A seed-specific promoter is appropriate forchanging the oil content of soybean seed. An endosperm-specific promoteris appropriate for changing the starch composition of corn seed. Aroot-specific promoter can be important for improving water or nutrientup-take in a plant. Control of expression of an introduced gene by thepromoter is important because it is sometimes detrimental to haveexpression of an introduced gene in non-target tissues. For example, agene which induces cell death can be expressed in male and/or femalegamete cells in connection with bioconfinement.

One of the primary goals of biotechnology is to obtain organisms, suchas plants, mammals, yeast, and prokaryotes having particular desiredcharacteristics or traits. Examples of these characteristics or traitsabound and may include, for example, in plants, virus resistance, insectresistance, herbicide resistance, enhanced stability or additionalnutritional value. Recent advances in genetic engineering have enabledresearchers in the field to incorporate polynucleotide sequences intohost cells to obtain the desired qualities in the organism of choice.This technology permits one or more polynucleotides from a sourcedifferent than the organism of choice to be transcribed by the organismof choice. If desired, the transcription and/or translation of these newpolynucleotides can be modulated in the organism to exhibit a desiredcharacteristic or trait. Alternatively, new patterns of transcriptionand/or translation of polynucleotides endogenous to the organism can beproduced.

SUMMARY OF THE INVENTION

The present invention is directed to isolated polynucleotide sequencesthat comprise promoters and promoter control elements from plants,especially Arabidopsis thaliana, and other promoters and promotercontrol elements functional in plants.

It is an object of the present invention to provide isolatedpolynucleotides that are promoter or promoter control sequences. Thesepromoter sequences comprise, for example,

-   -   (1) a polynucleotide having a nucleotide sequence according to        any one of SEQ. ID. Nos. 1-26 or residues 601-1000 of SEQ ID NO:        26;    -   (2) a polynucleotide having a nucleotide sequence having at        least 80% sequence identity to a sequence according to SEQ. ID.        Nos. 1-26 or residues 601-1000 of SEQ ID NO: 26; and    -   (3) a polynucleotide having a nucleotide sequence which        hybridizes to a sequence according to SEQ. ID. Nos. 1-26 or        nucleic acid residues 601-1000 of SEQ ID NO: 26 under a        condition establishing a Tm-5° C.

Promoter or promoter control element sequences of the present inventionare capable of modulating preferential transcription.

In another embodiment, the present promoter control elements are capableof serving as or fulfilling the function, for example, as a corepromoter, a TATA box, a polymerase binding site, an initiator site, atranscription binding site, an enhancer, an inverted repeat, a locuscontrol region, and/or a scaffold/matrix attachment region.

It is yet another object of the present invention to provide apolynucleotide that includes at least a first and a second promotercontrol element. The first promoter control element is a promotercontrol element sequence as discussed above, and the second promotercontrol element is heterologous to the first control element; wherein,the first and second control elements are operably linked. Suchpromoters may modulate transcript levels preferentially in a particulartissue or under particular conditions.

In another embodiment, the present isolated polynucleotide comprises apromoter or a promoter control element as described above, wherein thepromoter or promoter control element is operably linked to apolynucleotide to be transcribed.

In another embodiment of the present invention, the promoter andpromoter control elements of the instant invention are operably linkedto a heterologous polynucleotide that is a regulatory sequence.

It is another object of the present invention to provide a host cellcomprising an isolated polynucleotide or vector as described above orfragment thereof. Host cells include, for instance, bacterial, yeast,insect, mammalian, fungus, algae, and plant. The host cell can comprisea promoter or promoter control element exogenous to the genome. Such apromoter can modulate transcription in cis- and in trans-.

In yet another embodiment, the host cell is a plant cell capable ofregenerating into a plant.

It is yet another embodiment of the present invention to provide a plantcomprising an isolated polynucleotide or vector described above.

It is another object of the present invention to provide a method ofmodulating transcription in a sample that contains either a cell-freesystem of transcription or host cell. This method comprises providing apolynucleotide or vector according to the present invention as describedabove, and contacting the sample of the polynucleotide or vector withconditions that permit transcription.

In another embodiment of the present method, the polynucleotide orvector preferentially modulates, depending upon the function of theparticular promoter, constitutive transcription, stress inducedtranscription, light induced transcription, dark induced transcription,leaf transcription, root transcription, stem or shoot transcription,silique or fruit transcription, callus transcription, rhizometranscription, stem node transcription, gamete tissue transcription,flower transcription, immature bud and inflorescence specifictranscription, senescing induced transcription, germinationtranscription and/or drought transcription.

One embodiment of the invention is directed to an isolated nucleic acidmolecule having promoter activity comprising a nucleotide sequenceselected from the group consisting of:

-   -   a. a nucleotide sequence according to any one of SEQ ID NOs.        1-26;    -   b. a nucleotide sequence of nucleic acid residues 601-1000 of        SEQ ID NO: 26;    -   c. a nucleotide sequence comprising a functional fragment of (a)        or (b), wherein said fragment has promoter activity,        and wherein said isolated nucleic acid molecule is not SEQ ID        NO: 5.

Another embodiment of the invention is directed to an isolated nucleicacid molecule comprising a nucleotide sequence that shows at least 80percent sequence identity to any one of SEQ ID NOs: 1-26 or nucleic acidresidues 601-1000 of SEQ ID NO: 26, wherein said nucleic acid moleculecomprises a regulatory region that directs transcription of an operablylinked heterologous polynucleotide, and wherein said isolated nucleicacid molecule is not SEQ ID NO: 5.

In another embodiment of the invention the isolated nucleic acidmolecule shows at least 85 percent sequence identity to any one of SEQID NOs: 1-26 or nucleic acid residues 601-1000 of SEQ ID NO: 26.

In another embodiment of the invention the isolated nucleic acidmolecule has at least 90 percent sequence identity to any one of SEQ IDNOs: 1-26 or nucleic acid residues 601-1000 of SEQ ID NO: 26.

In another embodiment the isolated nucleic acid molecule comprises atleast one member selected from the group consisting of a promoter, anenhancer and an intron.

In a further embodiment of the invention, the isolated nucleic acidmolecule consists of any one of SEQ ID NOs: 1-4, 6-26 and the nucleicacid residues 601-1000 of SEQ ID NO: 26.

Another embodiment of the invention is directed to a vector constructcomprising:

a. a first nucleic acid molecule as described above; and

b. a transcribable polynucleotide molecule,

wherein said first nucleic acid molecule and said transcribablepolynucleotide molecule are heterologous to each other and are operablylinked.

In another embodiment of the invention, the first nucleic acid moleculeconsists of the nucleic acid molecule set forth in any one of SEQ IDNOs: 1-26 or nucleic acid residues 601-1000 of SEQ ID NO: 26.

In another embodiment of the invention, the transcribable polynucleotidemolecule encodes a polypeptide.

In another embodiment of the invention, the transcribable polynucleotidemolecule is operably linked to said first nucleic acid molecule in thesense orientation.

In another embodiment of the invention, the transcribable polynucleotidemolecule is transcribed into an RNA molecule that expresses thepolypeptide encoded by transcribable polynucleotide molecule.

In another embodiment of the invention, the transcribable polynucleotidemolecule is operably linked to said first nucleic acid molecule in theantisense orientation.

In another embodiment of the invention, the transcribable polynucleotidemolecule is transcribed into an antisense RNA molecule.

In another embodiment of the invention, the transcribable polynucleotidemolecule is transcribed into an interfering RNA against an endogenousgene.

In another embodiment of the invention, the transcribable polynucleotidemolecule encodes a polypeptide of agronomic interest.

Another embodiment of the invention is directed to a plant or plant cellcomprising:

-   -   a. the nucleic acid molecule described above that is operably        linked to a heterologous polynucleotide, or    -   b. the vector construct described above.

Another embodiment of the invention is directed to a plant or plant cellstably transformed with the vector construct described above.

Another embodiment of the invention is directed to a seed of a plant asdescribed above.

Another embodiment of the invention is directed to a method of directingtranscription by combining, in an environment suitable fortranscription:

a. a first nucleic acid molecule as described above; and

b. a transcribable polynucleotide molecule;

wherein said first nucleic acid molecule and said transcribablepolynucleotide molecule are heterologous to each other and operablylinked.

Another embodiment of the invention is directed to a method ofexpressing an exogenous coding region in a plant comprising:

-   -   a. transforming a plant cell with a vector as described above,    -   b. regenerating a stably transformed plant from the transformed        plant cell of step (a); and    -   c. selecting plants containing a transformed plant cell,        wherein expression of the transcribable polynucleotide molecule        results in production of a polypeptide encoded by said        transcribable polynucleotide molecule.

Another embodiment of the invention is directed to a method of alteringthe expression of a gene in a plant comprising:

-   -   a. transforming a plant cell with the nucleic acid molecule as        described above that is operably linked to a heterologous        polynucleotide, and    -   b. regenerating stably transformed plants from said transformed        plant cell.

Another embodiment of the invention is directed to a plant preparedaccording to the method described above.

Another embodiment of the invention is directed to a seed from the plantdescribed above.

Another embodiment of the invention is directed to a method of producinga transgenic plant, said method comprising;

a. introducing into a plant cell:

-   -   (i) an isolated polynucleotide comprising the nucleic acid as        described above that is operably linked to a heterologous        polynucleotide, or    -   (ii) the vector as described above; and

b. growing a plant from said plant cell.

Other and further objects of the present invention will be made clear orbecome apparent from the following description.

BRIEF DESCRIPTION OF THE TABLES AND FIGURES

The Tables consist of the Expression Reports for some of the promotersof the invention providing the nucleotide sequence for each promoter anddetails for expression driven by each of the nucleic acid promotersequences as observed in transgenic plants. The results are presented assummaries of the spatial expression, which provides information as togross and/or specific expression in various plant organs and tissues.The observed expression pattern is also presented, which gives detailsof expression during different generations or different developmentalstages within a generation. Additional information is provided regardingthe source organism of the promoter, and the vector and marker genesused for the construct. The following symbols are used consistentlythroughout the Tables:

T1: First generation transformant

T2: Second generation transformant

T3: Third generation transformant

(L): low expression level

(M): medium expression level

(H): high expression level

Each row of the table begins with heading of the data to be found in thesection. The following provides a description of the data to be found ineach section:

Heading in Tables Description 1. Promoter Expression Report # Identifiesthe particular promoter by its construct ID. 2. Promoter tested in:Identifies the organism in which the promoter- marker vector was tested.3. Spatial expression summary: Identifies the specific parts of theplant where various levels of GFP expression are observed. Expressionlevels are noted as either low (L), medium (M), or high (H). 4. Observedexpression pattern: Provides a general explanation of where GFPexpression in different generations of plants was observed. 5. Sourcepromoter organism: Identifies the plant species from which the promoterwas derived. 6. Vector: Identifies the vector used into which a promoterwas cloned. 7. Marker type: Identifies the type of marker linked to thepromoter. The marker is used to determine patterns of gene expression inplant tissue. 8. Generation screened: T1 Mature Identifies the plantgeneration(s) used in the T2 Seedling T2 Mature T3 screening process. T1plants are those plants Seedling subjected to the transformation eventwhile the T2 generation plants are from the seeds collected from the T1plants and T3 plants are from the seeds of T2 plants. 9. Inductionscompleted: Provides summary of experiment schedule. 10. T1 Mature PlantExpression: Identifies plant tissues that were observed for possibleexpression, and identifies (H, M or L) level of observed expression. 11.T2 Seedling Expression: Identifies plant tissues that were observed forpossible expression, and identifies (H, M or L) level of observedexpression. 12. T2 Mature Plant Expression: Identifies plant tissuesthat were observed for possible expression, and identifies (H, M or L)level of observed expression. 13. Utility Provides a description of theutility of the sequence, including a trait area and, in some instances,a sub-trait area. 14. Construct Identifies the promoter by its constructID and Promoter Candidate I.D. internal candidate number, and cDNAnumber cDNA I.D. 15. Lines/Events expressing: Identifies the line/eventnumbers that expressed under the promoter.Some promoter reports describe additional experiments and results withthe particular promoter.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of a vector pNewbin4-HAP1-GFP thatis useful to insert promoters of the invention into a plant. Thedefinitions of the abbreviations used in the vector map are as follows:

Ori—the origin of replication used by an E. coli host

RB—sequence for the right border of the T-DNA from pMOG800

BstXI—restriction enzyme cleavage site used for cloning

HAP1 VP16—coding sequence for a fusion protein of the HAP1 and VP16activation domains

NOS—terminator region from the nopaline synthase gene

HAP1UAS—the upstream activating sequence for HAP1

5ERGFP—the green fluorescent protein gene that has been optimized forlocalization to the endoplasmic reticulum

OCS2—the terminator sequence from the octopine synthase 2 gene

OCS—the terminator sequence from the octopine synthase gene

p28716 (a.k.a. 28716 short)—promoter used to drive expression of the PAT(BAR) gene

PAT (BAR)—a marker gene conferring herbicide resistance

LB—sequence for the left border of the T-DNA from pMOG800

Spec—a marker gene conferring spectinomycin resistance

TrfA—transcription repression factor gene

RK2-OriV—origin of replication for Agrobacterium

DETAILED DESCRIPTION OF THE INVENTION

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art.

The invention disclosed herein provides promoters capable of driving theexpression of an operably linked transgene. The design, construction,and use of these promoters is one object of this invention. The promotersequences, SEQ ID NOs: 1-26 and residues 601-1000 of SEQ ID NO: 26, arecapable of transcribing operably linked nucleic acid molecules inparticular plant tissues/organs or during particular plant growthstages, and therefore can selectively regulate expression of transgenesin these tissues/organs or at these times of plant development.

1. DEFINITIONS

Chimeric: The term “chimeric” is used to describe polynucleotides orgenes, or constructs wherein at least two of the elements of thepolynucleotide or gene or construct, such as the promoter and thepolynucleotide to be transcribed and/or other regulatory sequencesand/or filler sequences and/or complements thereof, are heterologous toeach other.

Broadly Expressing Promoter: Promoters referred to herein as “broadlyexpressing promoters” actively promote transcription under most, but notnecessarily all, environmental conditions and states of development orcell differentiation. Examples of broadly expressing promoters includethe cauliflower mosaic virus (CaMV) 35S transcript initiation region andthe 1′ or 2′ promoter derived from T-DNA of Agrobacterium tumefaciens,and other transcription initiation regions from various plant genes,such as the maize ubiquitin-1 promoter, known to those of skill.

Domain: Domains are fingerprints or signatures that can be used tocharacterize protein families and/or parts of proteins. Suchfingerprints or signatures can comprise conserved (1) primary sequence,(2) secondary structure, and/or (3) three-dimensional conformation. Asimilar analysis can be applied to polynucleotides. Generally, eachdomain has been associated with either a conserved primary sequence or asequence motif. Generally these conserved primary sequence motifs havebeen correlated with specific in vitro and/or in vivo activities. Adomain can be any length, including the entirety of the polynucleotideto be transcribed. Examples of domains include, without limitation, AP2,helicase, homeobox, zinc finger, etc.

Endogenous: The term “endogenous,” within the context of the currentinvention refers to any polynucleotide, polypeptide or protein sequencewhich is a natural part of a cell or organism(s) regenerated from saidcell. In the context of promoter, the term “endogenous coding region” or“endogenous cDNA” refers to the coding region that is naturally operablylinked to the promoter.

Enhancer/Suppressor: An “enhancer” is a DNA regulatory element that canincrease the steady state level of a transcript, usually by increasingthe rate of transcription initiation. Enhancers usually exert theireffect regardless of the distance, upstream or downstream location, ororientation of the enhancer relative to the start site of transcription.In contrast, a “suppressor” is a corresponding DNA regulatory elementthat decreases the steady state level of a transcript, again usually byaffecting the rate of transcription initiation. The essential activityof enhancer and suppressor elements is to bind a protein factor(s). Suchbinding can be assayed, for example, by methods described below. Thebinding is typically in a manner that influences the steady state levelof a transcript in a cell or in an in vitro transcription extract.

Exogenous: As referred to within, “exogenous” is any polynucleotide,polypeptide or protein sequence, whether chimeric or not, that isintroduced into the genome of a host cell or organism regenerated fromsaid host cell by any means other than by a sexual cross. Examples ofmeans by which this can be accomplished are described below, and includeAgrobacterium-mediated transformation (of dicots—e.g. Salomon et al.(1984) EMBO J. 3:141; Herrera-Estrella et al. (1983) EMBO J. 2:987; ofmonocots, representative papers are those by Escudero et al. (1996)Plant J. 10:355), Ishida et al. (1996) Nature Biotech 14:745, May et al.(1995) Bio/Technology 13:486), biolistic methods (Armaleo et al. (1990)Current Genetics 17:97), electroporation, in planta techniques, and thelike. Such a plant containing the exogenous nucleic acid is referred tohere as a T₀ for the primary transgenic plant and T₁ for the firstgeneration. The term “exogenous” as used herein is also intended toencompass inserting a naturally found element into a non-naturally foundlocation.

Heterologous sequences: “Heterologous sequences” are those that are notoperatively linked or are not contiguous to each other in nature. Forexample, a promoter from corn is considered heterologous to anArabidopsis coding region sequence. Also, a promoter from a geneencoding a growth factor from corn is considered heterologous to asequence encoding the corn receptor for the growth factor. Regulatoryelement sequences, such as UTRs or 3′ end termination sequences that donot originate in nature from the same gene as the coding sequence, areconsidered heterologous to said coding sequence. Elements operativelylinked in nature and contiguous to each other are not heterologous toeach other. On the other hand, these same elements remain operativelylinked but become heterologous if other filler sequence is placedbetween them. Thus, the promoter and coding sequences of a corn geneexpressing an amino acid transporter are not heterologous to each other,but the promoter and coding sequence of a corn gene operatively linkedin a novel manner are heterologous.

Homologous: In the current invention, a “homologous” polynucleotiderefers to a polynucleotide that shares sequence similarity with thepolynucleotide of interest. This similarity may be in only a fragment ofthe sequence and often represents a functional domain such as, examplesincluding, without limitation, a DNA binding domain or a domain withtyrosine kinase activity. The functional activities of homologouspolynucleotides are not necessarily the same.

Inducible Promoter: An “inducible promoter” in the context of thecurrent invention refers to a promoter, the activity of which isinfluenced by certain conditions, such as light, temperature, chemicalconcentration, protein concentration, conditions in an organism, cell,or organelle, etc. A typical example of an inducible promoter, which canbe utilized with the polynucleotides of the present invention, isPARSK1, the promoter from an Arabidopsis gene encoding aserine-threonine kinase enzyme, and which promoter is induced bydehydration, abscisic acid and sodium chloride (Wang and Goodman (1995)Plant J. 8:37). Examples of environmental conditions that may affecttranscription by inducible promoters include anaerobic conditions,elevated temperature, the presence or absence of a nutrient or otherchemical compound or the presence of light.

Misexpression: The term “misexpression” refers to an increase or adecrease in the transcription of a coding region into a complementaryRNA sequence as compared to the wild-type. This term also encompassesexpression and/or translation of a gene or coding region or inhibitionof such transcription and/or translation for a different time period ascompared to the wild-type and/or from a non-natural location within theplant genome, including a gene or coding region from a different plantspecies or from a non-plant organism.

Modulate Transcription Level: As used herein, the phrase “modulatetranscription” describes the biological activity of a promoter sequenceor promoter control element. Such modulation includes, withoutlimitation, up- and down-regulation of initiation of transcription, rateof transcription, and/or transcription levels.

Operable Linkage: An “operable linkage” is a linkage in which a promotersequence or promoter control element is connected to a polynucleotidesequence (or sequences) in such a way as to place transcription of thepolynucleotide sequence under the influence or control of the promoteror promoter control element. Two DNA sequences (such as a polynucleotideto be transcribed and a promoter sequence linked to the 5′ end of thepolynucleotide to be transcribed) are said to be operably linked ifinduction of promoter function results in the transcription of mRNAencoding the polynucleotide and if the nature of the linkage between thetwo DNA sequences does not (1) result in the introduction of aframe-shift mutation, (2) interfere with the ability of the promotersequence to direct the expression of the protein, antisense RNA, RNAi orribozyme, or (3) interfere with the ability of the DNA template to betranscribed. Thus, a promoter sequence would be operably linked to apolynucleotide sequence if the promoter was capable of effectingtranscription of that polynucleotide sequence.

Percentage of sequence identity As used herein, the term “percentsequence identity” refers to the degree of identity between any givenquery sequence, e.g., SEQ ID NOs:1-26, and a subject sequence. A subjectsequence typically has a length that is from about 80 percent to 250percent of the length of the query sequence, e.g., 82, 85, 87, 89, 90,93, 95, 97, 99, 100, 105, 110, 115, or 120, 130, 140, 150, 160, 170,180, 190, 200, 210, 220, 230, 240, or 250 percent of the length of thequery sequence. A query nucleic acid or amino acid sequence is alignedto one or more subject nucleic acid or amino acid sequences using thecomputer program ClustalW (version 1.83, default parameters), whichallows alignments of nucleic acid or protein sequences to be carried outacross their entire length (global alignment). Chenna et al. (2003)Nucleic Acids Res. 31(13):3497-500.

ClustalW calculates the best match between a query and one or moresubject sequences, and aligns them so that identities, similarities anddifferences can be determined. Gaps of one or more residues can beinserted into a query sequence, a subject sequence, or both, to maximizesequence alignments. For fast pairwise alignment of nucleic acidsequences, the following default parameters are used: word size: 2;window size: 4; scoring method: percentage; number of top diagonals: 4;and gap penalty: 5. For an alignment of multiple nucleic acid sequences,the following parameters are used: gap opening penalty: 10.0; gapextension penalty: 5.0; and weight transitions: yes. For fast pairwisealignment of protein sequences, the following parameters are used: wordsize: 1; window size: 5; scoring method: percentage; number of topdiagonals: 5; gap penalty: 3. For multiple alignment of proteinsequences, the following parameters are used: weight matrix: blosum; gapopening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps:on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, andLys; residue-specific gap penalties: on. The output is a sequencealignment that reflects the relationship between sequences. ClustalW canbe run, for example, at the Baylor College of Medicine Search Launcherwebsite and at the European Bioinformatics Institute website on theWorld Wide Web.

To determine a percent identity of a subject polypeptide or nucleic acidsequence to a query sequence, the sequences are aligned using Clustal W,the number of identical matches in the alignment is divided by thelength of the query sequence, and the result is multiplied by 100. Theoutput is the percent identity of the subject sequence with respect tothe query sequence. It is noted that the percent identity value can berounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and78.19 are rounded up to 78.2.

Plant Promoter: A “plant promoter” is a promoter capable of initiatingtranscription in plant cells and can modulate transcription of apolynucleotide. Such promoters need not be of plant origin. For example,promoters derived from plant viruses, such as the CaMV35S promoter orfrom Agrobacterium tumefaciens such as the T-DNA promoters, can be plantpromoters. A typical example of a plant promoter of plant origin is themaize ubiquitin-1 (ubi-1) promoter known to those of skill in the art.

Plant Tissue: The term “plant tissue” includes differentiated andundifferentiated tissues or plants, including but not limited to roots,stems, shoots, rhizomes, cotyledons, epicotyl, hypocotyl, leaves,pollen, seeds, gall tissue and various forms of cells in culture such assingle cells, protoplast, embryos, and callus tissue. The plant tissuemay be in plants or in organ, tissue or cell culture.

Preferential Transcription: “Preferential transcription” is defined astranscription that occurs in a particular pattern of cell types ordevelopmental times or in response to specific stimuli or combinationthereof. Non-limitive examples of preferential transcription include:high transcript levels of a desired sequence in root tissues; detectabletranscript levels of a desired sequence in certain cell types duringembryogenesis; and low transcript levels of a desired sequence underdrought conditions. Such preferential transcription can be determined bymeasuring initiation, rate, and/or levels of transcription.

Promoter: A “promoter” is a DNA sequence that directs the transcriptionof a polynucleotide. Typically a promoter is located in the 5′ region ofa polynucleotide to be transcribed, proximal to the transcriptionalstart site of such polynucleotide. More typically, promoters are definedas the region upstream of the first exon; more typically, as a regionupstream of the first of multiple transcription start sites; moretypically, as the region downstream of the preceding gene and upstreamof the first of multiple transcription start sites; more typically, theregion downstream of the polyA signal and upstream of the first ofmultiple transcription start sites; even more typically, about 3,000nucleotides upstream of the ATG of the first exon; even more typically,2,000 nucleotides upstream of the first of multiple transcription startsites. The promoters of the invention comprise at least a core promoteras defined above. Frequently promoters are capable of directingtranscription of genes located on each of the complementary DNA strandsthat are 3′ to the promoter. Stated differently, many promoters exhibitbidirectionality and can direct transcription of a downstream gene whenpresent in either orientation (i.e. 5′ to 3′ or 3′ to 5′ relative to thecoding region of the gene). Additionally, the promoter may also includeat least one control element such as an upstream element. Such elementsinclude UARs and optionally, other DNA sequences that affecttranscription of a polynucleotide such as a synthetic upstream element.

Promoter Control Element: The term “promoter control element” as usedherein describes elements that influence the activity of the promoter.Promoter control elements include transcriptional regulatory sequencedeterminants such as, but not limited to, enhancers, scaffold/matrixattachment regions, TATA boxes, transcription start locus controlregions, UARs, URRs, other transcription factor binding sites andinverted repeats.

Public sequence: The term “public sequence,” as used in the context ofthe instant application, refers to any sequence that has been depositedin a publicly accessible database prior to the filing date of thepresent application. This term encompasses both amino acid andnucleotide sequences. Such sequences are publicly accessible, forexample, on the BLAST databases on the NCBI FTP web site (accessible viathe interact). The database at the NCBI FTP site utilizes “gi” numbersassigned by NCBI as a unique identifier for each sequence in thedatabases, thereby providing a non-redundant database for sequence fromvarious databases, including GenBank, EMBL, DBBJ (DNA Database of Japan)and PDB (Brookhaven Protein Data Bank).

Regulatory Regions: The term “regulatory region” refers to nucleotidesequences that, when operably linked to a sequence, influencetranscription initiation or translation initiation or transcriptiontermination of said sequence and the rate of said processes, and/orstability and/or mobility of a transcription or translation product. Asused herein, the term “operably linked” refers to positioning of aregulatory region and said sequence to enable said influence. Regulatoryregions include, without limitation, promoter sequences, enhancersequences, response elements, protein recognition sites, inducibleelements, protein binding sequences, 5′ and 3′ untranslated regions(UTRs), transcriptional start sites, termination sequences,polyadenylation sequences, and introns.

The nucleic acid sequence set forth in SEQ ID NOs:1-26 are examples ofregulatory regions provided herein. However, a regulatory region canhave a nucleotide sequence that deviates from that set forth in SEQ IDNOs:1-26, while retaining the ability to direct expression of anoperably linked nucleic acid. For example, a regulatory region having80% or greater (e.g. 85% or greater, 90% or greater 91% or greater, 92%or greater, 93% or greater, 94% or greater, 95% or greater, 96% orgreater, 97% or greater, 98% or greater, or 99% or greater) sequenceidentity to the nucleotide sequence set forth in SEQ ID NOs:1-25, or 26can direct expression of an operably linked nucleic acid.

A regulatory region can also be a fragment of SEQ ID NOs:1-25, or 26,while retaining promoter activity, i.e. the ability to direct expressionof an operably linked nucleic acid. Additional examples of regulatoryregions are identified in the Sequence Listing.

Regulatory regions can be classified in two categories, promoters andother regulatory regions.

Regulatory Sequence: The term “regulatory sequence,” as used in thecurrent invention, refers to any nucleotide sequence that influencestranscription or translation initiation and rate, or stability and/ormobility of a transcript or polypeptide product. Regulatory sequencesinclude, but are not limited to, promoter sequences, enhancer sequences,response elements, protein recognition sites, inducible elements,promoter control elements, protein binding sequences, 5′ and 3′untranslated regions (UTRs), transcriptional start sites, terminationsequences, polyadenylation sequences, introns, certain sequences withinamino acid coding sequences such as secretory signals, protease cleavagesites, etc.

A 5′ untranslated region (5′ UTR) of a gene is generally defined as apolynucleotide segment between the transcription start site (TSS) andthe coding sequence start site (ATG codon) of a messenger RNA or cDNA.Alternately, 5′ UTR can be synthetically produced or manipulated DNAelements. A “plant 5′UTR” can be a native or non-native 5′UTR that isfunctional in plant cells. A 5′ UTR can be used as a 5′ regulatoryelement for modulating expression of an operably linked transcribablepolynucleotide molecule. For example, 5′ UTRs derived from heat shockprotein genes have been demonstrated to enhance gene expression inplants (see for example, U.S. Pat. No. 5,659,122 and U.S. Pat. No.5,362,865, all of which are incorporated herein by reference). Examplesof 5′UTRs include those shown in SEQ ID NOs: 1-4, 6-10, 13-26.

Specific Promoters: In the context of the current invention, “specificpromoters” refers to a subset of promoters that have a high preferencefor modulating transcript levels in a specific tissue, or organ or celland/or at a specific time during development of an organism. By “highpreference” is meant at least 3-fold, preferably 5-fold, more preferablyat least 10-fold still more preferably at least 20-fold, 50-fold or100-fold increase in transcript levels under the specific condition overthe transcription under any other reference condition considered.Typical examples of temporal and/or tissue or organ specific promotersof plant origin that can be used with the polynucleotides of the presentinvention, are: PTA29, a promoter which is capable of driving genetranscription specifically in tapetum and only during anther development(Koltonow et al. (1990) Plant Cell 2:1201; RCc2 and RCc3, promoters thatdirect root-specific gene transcription in rice (Xu et al. (1995) PlantMol. Biol. 27:237; TobRB27, a root-specific promoter from tobacco(Yamamoto et al. (1991) Plant Cell 3:371). Examples of tissue-specificpromoters under developmental control include promoters that initiatetranscription only in certain tissues or organs, such as root, ovule,fruit, seeds, or flowers. Other specific promoters include those fromgenes encoding seed storage proteins or the lipid body membrane protein,oleosin. A few root-specific promoters are noted above. See also“Preferential transcription.”

A regulatory region can contain conserved regulatory motifs. Such aregulatory region can be any one of the sequences set forth in SEQ IDNOs:1-26, or a regulatory region having a nucleotide sequence thatdeviates from any one of those set forth in SEQ ID NOs:1-26, whileretaining the ability to direct expression of an operably linked nucleicacid. For example, a regulatory region can contain a CAAT box or a TATAbox. A CAAT box is a conserved nucleotide sequence involved ininitiation of transcription. A CAAT box functions as a recognition andbinding site for regulatory proteins called transcription factors. ATATA box is another conserved nucleotide sequence involved intranscription initiation. A TATA box seems to be important indetermining accurately the position at which transcription is initiated.

Other conserved regulatory motifs can be identified using methods knownin the art. For example, a regulatory region can be analyzed using thePLACE (PLAnt Cis-acting regulatory DNA Elements) Web Signal Scan programon the world wide web at dna.affrc.go.jp/PLACE/signalscan.html. See,Higo et al., Nucleic Acids Research, 27(1):297-300 (1999); andPrestridge, CABIOS, 7:203-206 (1991). Examples of conserved regulatorymotifs can be found in the PLACE database on the world wide web atdna.affrc.go.jp/PLACE/. See, Higo et al., supra.

A regulatory region such as any one of SEQ ID NOs:1-26, or a regulatoryregion having a nucleotide sequence that deviates from those set forthin SEQ ID NOs:1-26, while retaining the ability to direct expression ofan operably linked nucleic acid, can contain one or more conservedregulatory motifs, which can be found in the PLACE database. Forexample, such a regulatory region can contain a −300CORE motif havingthe consensus sequence TGTAAAG. See, Forde et al., Nucleic Acids Res13:7327-7339 (1985); Colot et al., EMBO J 6:3559-3564 (1987); Thomas andFlavell, Plant Cell 2:1171-1180 (1990); Thompson et al., Plant Mol Biol15:755-764 (1990); Vicente-Carbajosa et al., Proc Natl Acad Sci USA94:7685-7690 (1997); Mena et al., Plant J 16:53-62 (1998); Shing, PlantPhysiol 118: 1111-1120 (1998). Such a regulatory region can contain anABREATCONSENSUS motif having the consensus sequence YACGTGGC. See, Choiet al., J Biol Chem 275: 1723-1730 (2000); Kang et al., Plant Cell 14:343-357 (2002); Oh et al., Plant Physiology 138: 341-351 (2005); Choi etal., Plant Physiol 139: 1750-1761 (2005). Such a regulatory region cancontain an ABREATRD22 motif having the consensus sequence RYACGTGGYR.See, Iwasaki et al., Mol Gen Genet 247:391-398 (1995); Bray, Trends inPlant Science 2:48-54 (1997); Busk and Pages, Plant Mol Biol 37:425-435(1998). A regulatory region can contain an ABRELATERD1 motif having theconsensus sequence ACGTG. See, Simpson et al., Plant J 33: 259-270(2003); Nakashima et al., Plant Mol Biol 60:51-68 (2006). A regulatoryregion can contain an ABREMOTIFAOSOSEM motif having the consensussequence TACGTGTC. See, Hattori et al., Plant J 7: 913-925 (1995); Hoboet al., Proc Natl Acad Sci USA 96: 15348-15353 (1999). A regulatoryregion can contain an ABRERATCAL motif having the consensus sequenceMACGYGB. See, Kaplan et al., Plant Cell 18:2733-2748 (2006). Aregulatory region can contain an ACGTCBOX motif having the consensussequence GACGTC. See, Foster et al., FASEB J 8:192-200 (1994); Izawa etal., Plant Cell 6:1277-1287 (1994); Izawa et al., J Mol Biol230:1131-1144 (1993). A regulatory region can contain an ACGTOSGLUB1motif having the consensus sequence GTACGTG. See, Washida et al., PlantMol Biol 40:1-12 (1999); Wu et al., Plant J 23: 415-421 (2000). Aregulatory region can contain an ACGTTBOX motif having the consensussequence AACGTT. See, Foster et al., FASEB J 8:192-200 (1994). Aregulatory region can contain an ACIIPVPAL2 motif having the consensussequence CCACCAACCCCC. See, Patzlaff et al., Plant Mol Biol 53:597-608(2003); Hatton et al., Plant J 7:859-876 (1995); Gomez-Maldonado et al.,Plant J 39:513-526 (2004). A regulatory region can contain anAGL2ATCONSENSUS motif having the consensus sequence NNWNCCAWWWWTRGWWAN.See, Huang et al., Plant Cell 8: 81-94 (1996). A regulatory region cancontain an AMYBOX2 motif having the consensus sequence TATCCAT. See,Huang et al., Plant Mol Biol 14:655-668 (1990); Hwang et al., Plant MolBiol 36:331-341 (1998). A regulatory region can contain anANAERO1CONSENSUS motif having the consensus sequence AAACAAA. See,Mohanty et al., Ann Bot (Lond). 96: 669-681 (2005). A regulatory regioncan contain an ARE1 motif having the consensus sequence RGTGACNNNGC.See, Rushmore et al., J Biol Chem 266:11632-11639 (1991). A regulatoryregion can contain an ATHB6COREAT motif having the consensus sequenceCAATTATTA. See, Himmelbach et al., EMBO J 21:3029-3038 (2002). Aregulatory region can contain an AUXRETGA1GMGH3 motif having theconsensus sequence TGACGTAA. See, Liu et al., Plant Cell 6:645-657(1994); Liu et al., Plant Physiol 115:397-407 (1997); Guilfoyle et al.,Plant Physiol 118: 341-347 (1998). A regulatory region can contain aBOXIIPCCHS motif having the consensus sequence ACGTGGC. See, Block etal., Proc Natl Acad Sci USA 87:5387-5391 (1990); Terzaghi and Cashmore,Annu Rev Plant Physiol Plant Mol Biol 46:445-474 (1995); Nakashima etal., Plant Mol Biol 60: 51-68 (2006). A regulatory region can contain aBOXLCOREDCPAL motif having the consensus sequence ACCWWCC. See, Meada etal., Plant Mol Biol 59: 739-752. (2005). A regulatory region can containa CACGCAATGMGH3 motif having the consensus sequence CACGCAAT. See,Ulmasov et al., Plant Cell 7: 1611-1623 (1995). A regulatory region cancontain a CARGATCONSENSUS motif having the consensus sequenceCCWWWWWWGG. See, Hepworth et al., EMBO J 21: 4327-4337 (2002); Michaelset al., Plant J 33: 867-874 (2003); Hong et al., Plant Cell 15:1296-1309(2003); Folter and Angenent, Trends Plant Sci 11:224-231 (2006). Aregulatory region can contain a CARGCW8GAT motif having the consensussequence CWWWWWWWWG. See, Tang and Perry, J Biol Chem 278:28154-28159(2003); Folter and Angenent, Trends Plant Sci 11:224-231 (2006). Aregulatory region can contain a CIACADIANLELHC motif having theconsensus sequence CAANNNNATC. See, Piechulla et al., Plant Mol Biol38:655-662 (1998). A regulatory region can contain a DPBFCOREDCDC3 motifhaving the consensus sequence ACACNNG. See, Kim et al., Plant J 11:1237-1251 (1997); Finkelstein and Lynch, Plant Cell 12: 599-609 (2000);Lopez-Molina and Chua, Plant Cell Physiol 41: 541-547 (2000). Aregulatory region can contain a DRE2COREZMRAB17 motif having theconsensus sequence ACCGAC. See, Busk et al., Plant J 11: 1285-1295(1997); Dubouzet et al., Plant J 33: 751-763 (2003); Kizis and Pages,Plant J 30 :679-689 (2002). A regulatory region can contain anE2FCONSENSUS motif having the consensus sequence WTTSSCSS. See,Vandepoele et al., Plant Physiol 139: 316-328. (2005). A regulatoryregion can contain an EMHVCHORD motif having the consensus sequenceTGTAAAGT. See, Muller and Knudsen, Plant J 4: 343-355 (1993). Aregulatory region can contain an EVENINGAT motif having the consensussequence AAAATATCT. See, Rawat et al., Plant Mol Biol 57: 629-643 (2005)and Harmer et al., Science 290: 2110-2113 (2000). A regulatory regioncan contain an GLMHVCHORD motif having the consensus sequence RTGASTCAT.See, Albani et al., Plant Cell 9: 171-184 (1997); Muller M Plant J 4:343-355 (1993); Onate et al., J Biol Chem 274: 9175-9182 (1999). Aregulatory region can contain a GT1 Consensus motif having the consensussequence GRWAAW. See, Terzaghi and Cashmore, supra.; Villain et al., JBiol Chem 271:32593-32598 (1996); Le Gourrierec et al., Plant J18:663-668 (1999); Buchel et al., Plant Mol Biol 40:387-396 (1999);Zhou, Trends in Plant Science 4:210-214 (1999). A regulatory region cancontain a GT1GMSCAM4 motif having the consensus sequence GAAAAA. See,Park et al., Plant Physiol 135: 2150-2161 (2004). A regulatory regioncan contain a HDZIP2ATATHB2 motif having the consensus sequenceTAATMATTA. See, Ohgishi et al., Plant J 25: 389-398 (2001). A regulatoryregion can contain an IBOXCORENT motif having the consensus sequenceGATAAGR. See, Martinez-Hernandez et al., Plant Physiol 128:1223-1233(2002). A regulatory region can contain an INRNTPSADB motif having theconsensus sequence YTCANTYY. See, Nakamura et al., Plant J 29: 1-10(2002). A regulatory region can contain a LEAFYATAG motif having theconsensus sequence CCAATGT. See, Kamiya et al., Plant J 35: 429-441(2003). A regulatory region can contain a LRENPCABE motif having theconsensus sequence ACGTGGCA. See, Castresana et al., EMBO J 7:1929-1936(1988). A regulatory region can contain a MARTBOX motif having theconsensus sequence TTWTWTTWTT. See, Gasser et al., Intnatl Rev Cyto119:57-96 (1989). A regulatory region can contain a MYBGAHV motif havingthe consensus sequence TAACAAA. See, Gubler et al., Plant Cell7:1879-1891 (1995); Morita et al., FEBS Lett 423:81-85 (1998); Gubler etal., Plant J 17:1-9 (1999). A regulatory region can contain a MYBPLANTmotif having the consensus sequence MACCWAMC. See, Sablowski et al.,EMBO J 13:128-137 (1994); Tamagnone et al., Plant Cell 10: 135-154(1998). A regulatory region can contain a NRRBNEXTA motif having theconsensus sequence TAGTGGAT. See, Elliott and Shirsat, Plant Mol Biol37:675-687 (1998). A regulatory region can contain an O2F3BE2S1 motifhaving the consensus sequence TCCACGTACT. See, Vincentz et al., PlantMol Biol 34:879-889 (1997). A regulatory region can contain a P1BS motifhaving the consensus sequence GNATATNC. See, Rubio et al., Genes Dev.15: 2122-2133. (2001); Shunmann et al., J Exp Bot. 55: 855-865. (2004);Shunmann et al., Plant Physiol 136: 4205-4214. (2004). A regulatoryregion can contain a PRECONSCRHSP70A motif having the consensus sequenceSCGAYNRNNNNNNNNNNNNNNHD. See, von Gromoff et al., Nucleic Acids Res34:4767-4779 (2006). A regulatory region can contain a PROXBBNNAPA motifhaving the consensus sequence CAAACACC. See, Ezcurra et al., Plant MolBiol 40:699-709 (1999); Busk and Pages, supra.; Ezcurra et al., Plant J24:57-66 (2000). A regulatory region can contain a PYRIMIDINEBOXHVEPB1motif having the consensus sequence TTTTTTCC. See, Cercos et al., PlantJ 19: 107-118 (1999). A regulatory region can contain a RBCSCONSENSUSmotif having the consensus sequence AATCCAA. See, Manzara and Gruissem,Photosynth Res 16:117-139 (1988); Donald and Cashmore, EMBO J9:1717-1726 (1990). A regulatory region can contain a ROOTMOTIFTAPDX1motif having the consensus sequence ATATT. See, Elmayan and Tepfer,Transgenic Res 4:388-396 (1995). A regulatory region can contain aRYREPEATVFLEB4 motif having the consensus sequence CATGCATG. See, Curabaet al., Plant Physiol 136: 3660-3669. (2004); Nag et al., Plant Mol Biol59: 821-838 (2005). A regulatory region can contain a SEF1MOTIF motifhaving the consensus sequence ATATTTAWW. See, Allen et al., Plant Cell1:623-631 (1989); Lessard et al., Plant Mol Biol 16:397-413 (1991). Aregulatory region can contain a SORLREP3AT motif having the consensussequence TGTATATAT. See, Hudson and Quail, Plant Physiol 133: 1605-1616(2003). A regulatory region can contain a SURE2STPAT21 motif having theconsensus sequence AATACTAAT. See, Grierson et al., Plant J 5:815-826(1994). A regulatory region can contain a SV40COREENHAN motif having theconsensus sequence GTGGWWHG. See, Weiher et al., Science 219:626-631(1983); Green et al., EMBO J 6:2543-2549 (1987); Donald and Cashmore,EMBO J 9:1717-1726 (1990). A regulatory region can contain a TATABOX2motif having the consensus sequence TATAAAT. See, Shirsat et al., MolGen Genet 215:326-331 (1989); Grace et al., Biol Chem 279:8102-8110(2004). A regulatory region can contain a TATABOX3 motif having theconsensus sequence TATTAAT. See, PLACE (PLAnt Cis-acting regulatory DNAElements) at dna.affrc.go.jp/PLCAE/signalscan.html). A regulatory regioncan contain a TATABOX4 motif having the consensus sequence TATATAA. See,Grace et al., J Biol Chem 279:8102-8110 (2004). A regulatory region cancontain a TATABOX5 motif having the consensus sequence TTATTT. See,Tjaden et al., Plant Physiol 108:1109-1117 (1995). A regulatory regioncan contain a TATABOXOSPAL motif having the consensus sequence TATTTAA.See, Zhu et al., Plant Cell 14: 795-803 (2002). A regulatory region cancontain a TELOBOXATEEF1AA1 motif having the consensus sequenceAAACCCTAA. See, Tremousayque et al., Plant J 20: 553-561 (1999); Axeloset al., Mol Gen Genet 219: 106-112 (1989); Welchen and Gonzalez, PlantPhysiol 139: 88-100 (2005). A regulatory region can contain a TL1ATSARmotif having the consensus sequence CTGAAGAAGAA. See, Wang et al.,Science 308: 1036-1040 (2005). A regulatory region can contain aUP2ATMSD motif having the consensus sequence AAACCCTA. See, Tatematsu etal., Plant Physiology 138: 757-766 (2005). A regulatory region cancontain a WBBOXPCWRKY1 motif having the consensus sequence TTTGACY. See,Ishiguro and Nakamura, Mol Gen Genet 244:563-571 (1994); Rushton et al.,Plant Mol Biol 29:691-702 (1995); Rushon et al., EMBO J 15:5690-5700(1996); de Pater et al., Nucleic Acids Res 24:4624-4631 (1996); Eulgemet al., Trends Plant Sci 5: 199-206 (2000). A regulatory region cancontain a XYLAT motif having the consensus sequence ACAAAGAA. See, Ko etal., Mol Genet Genomics 276:517-531 (2006)

Stringency: “Stringency,” as used herein is a function of nucleic acidmolecule probe length, nucleic acid molecule probe composition (G+Ccontent), salt concentration, organic solvent concentration andtemperature of hybridization and/or wash conditions. Stringency istypically measured by the parameter T_(m), which is the temperature atwhich 50% of the complementary nucleic acid molecules in thehybridization assay are hybridized, in terms of a temperaturedifferential from T_(m). High stringency conditions are those providinga condition of T_(m)-5° C. to T_(m)-10° C. Medium or moderate stringencyconditions are those providing T_(m)-20° C. to T_(m)-29° C. Lowstringency conditions are those providing a condition of T_(m)-40° C. toT_(m)-48° C. The relationship between hybridization conditions and T_(m)(in ° C.) is expressed in the mathematical equation:

T _(m)=81.5−16.6(log₁₀[Na⁺])+0.41(%G+C)−(600/N)  (I)

where N is the number of nucleotides of the nucleic acid molecule probe.This equation works well for probes 14 to 70 nucleotides in length thatare identical to the target sequence. The equation below, for T_(m) ofDNA-DNA hybrids, is useful for probes having lengths in the range of 50to greater than 500 nucleotides, and for conditions that include anorganic solvent (formamide):

T _(m)=81.5+16.6 log {[Na⁺]/(1+0.7[Na⁺])}+0.41(%G+C)−500/L0.63(%formamide)  (II)

where L represents the number of nucleotides in the probe in the hybrid(21). The T_(m) of Equation II is affected by the nature of the hybrid:for DNA-RNA hybrids, T_(m) is 10-15° C. higher than calculated; forRNA-RNA hybrids, T_(m) is 20-25° C. higher. Because the T_(m) decreasesabout 1° C. for each 1% decrease in homology when a long probe is used(Frischauf et al. (1983) J. Mol Biol, 170: 827-842), stringencyconditions can be adjusted to favor detection of identical genes orrelated family members.

Equation II is derived assuming the reaction is at equilibrium.Therefore, hybridizations according to the present invention are mostpreferably performed under conditions of probe excess and allowingsufficient time to achieve equilibrium. The time required to reachequilibrium can be shortened by using a hybridization buffer thatincludes a hybridization accelerator such as dextran sulfate or anotherhigh volume polymer.

Stringency can be controlled during the hybridization reaction, or afterhybridization has occurred, by altering the salt and temperatureconditions of the wash solutions. The formulas shown above are equallyvalid when used to compute the stringency of a wash solution. Preferredwash solution stringencies lie within the ranges stated above; highstringency is 5-8° C. below T_(m), medium or moderate stringency is26-29° C. below T_(m) and low stringency is 45-48° C. below T_(m).

T₀: The term “T₀” refers to the whole plant, explant or callus tissue,inoculated with the transformation medium.

T₁: The term T₁ refers to either the progeny of the T₀ plant, in thecase of whole-plant transformation, or the regenerated seedling in thecase of explant or callous tissue transformation.

T₂: The term T₂ refers to the progeny of the T₁ plant. T₂ progeny arethe result of self-fertilization or cross-pollination of a T₁ plant.

T₃: The term T₃ refers to second generation progeny of the plant that isthe direct result of a transformation experiment. T₃ progeny are theresult of self-fertilization or cross-pollination of a T₂ plant.

TATA to start: “TATA to start” shall mean the distance, in number ofnucleotides, between the primary TATA motif and the start oftranscription.

Transgenic plant: A “transgenic plant” is a plant having one or moreplant cells that contain at least one exogenous polynucleotideintroduced by recombinant nucleic acid methods.

Translational start site: In the context of the present invention, a“translational start site” is usually an ATG or AUG in a transcript,often the first ATG or AUG. A single protein encoding transcript,however, may have multiple translational start sites.

Transcription start site: “Transcription start site” is used in thecurrent invention to describe the point at which transcription isinitiated. This point is typically located about 25 nucleotidesdownstream from a TFIID binding site, such as a TATA box. Transcriptioncan initiate at one or more sites within the gene, and a singlepolynucleotide to be transcribed may have multiple transcriptional startsites, some of which may be specific for transcription in a particularcell-type or tissue or organ. “+1” is stated relative to thetranscription start site and indicates the first nucleotide in atranscript.

Upstream Activating Region (UAR): An “Upstream Activating Region” or“UAR” is a position or orientation dependent nucleic acid element thatprimarily directs tissue, organ, cell type, or environmental regulationof transcript level, usually by affecting the rate of transcriptioninitiation. Corresponding DNA elements that have a transcriptioninhibitory effect are called herein “Upstream Repressor Regions” or“URR”s. The essential activity of these elements is to bind a proteinfactor. Such binding can be assayed by methods described below. Thebinding is typically in a manner that influences the steady state levelof a transcript in a cell or in vitro transcription extract.

Untranslated region (UTR): A “UTR” is any contiguous series ofnucleotide bases that is transcribed, but is not translated. A 5′ UTRlies between the start site of the transcript and the translationinitiation codon and includes the +1 nucleotide. A 3′ UTR lies betweenthe translation termination codon and the end of the transcript. UTRscan have particular functions such as increasing mRNA message stabilityor translation attenuation. Examples of 3′ UTRs include, but are notlimited to polyadenylation signals and transcription terminationsequences.

2. USE OF THE PROMOTERS OF THE INVENTION

The promoters and promoter control elements of this invention arecapable of modulating transcription. Such promoters and promoter controlelements can be used in combination with native or heterologous promoterfragments, control elements or other regulatory sequences to modulatetranscription and/or translation.

Specifically, promoters and control elements of the invention can beused to modulate transcription of a desired polynucleotide, whichincludes without limitation:

-   -   (i) antisense;    -   (ii) ribozymes;    -   (iii) coding sequences; or    -   (iv) fragments thereof.

The promoter also can modulate transcription in a host genome in cis- orin trans-.

In an organism, such as a plant, the promoters and promoter controlelements of the instant invention are useful to produce preferentialtranscription which results in a desired pattern of transcript levels ina particular cells, tissues, or organs, or under particular conditions.

4. IDENTIFYING AND ISOLATING PROMOTER SEQUENCES OF THE INVENTION

The promoters and promoter control elements of the present invention arepresented in the Promoter Reports of the Tables and were identified fromArabidopsis thaliana and Oryza sativa. Isolation from genomic librariesof polynucleotides comprising the sequences of the promoters andpromoter control elements of the present invention is possible usingknown techniques. For example, polymerase chain reaction (PCR) canamplify the desired polynucleotides utilizing primers designed from SEQID NOs: 1-26 or residues 601-1000 of SEQ ID NO: 26. Polynucleotidelibraries comprising genomic sequences can be constructed according toSambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) Ed.(1989) Cold Spring Harbor Press, Cold Spring Harbor, N.Y.), for example.

Other procedures for isolating polynucleotides comprising the promotersequences of the invention include, without limitation, tail-PCR, and 5′rapid amplification of cDNA ends (RACE). See, for tail-PCR, for example,Liu et al. (1995) Plant J 8(3): 457-463; Liu et al. (1995) Genomics 25:674-681; Liu et al. (1993) Nucl. Acids Res. 21(14): 3333-3334; and Zoeet al. (1999) BioTechniques 27(21: 240-248; for RACE, see, for example,PCR Protocols: A Guide to Methods and Applications, (1990) AcademicPress, Inc.

In addition, the promoters and promoter control elements described inthe Promoter Reports in the Tables (SEQ. ID. Nos. 1-26) can bechemically synthesized according to techniques in common use. See, forexample, Beaucage et al. (1981) Tet. Lett. 22: 1859 and U.S. Pat. No.4,668,777. Such chemical oligonucleotide synthesis can be carried outusing commercially available devices, such as, Biosearch 4600 or 8600DNA synthesizer, by Applied Biosystems, a division of Perkin-ElmerCorp., Foster City, Calif., USA; and Expedite by Perceptive Biosystems,Framingham, Mass., USA.

Included in the present invention are promoters exhibiting nucleotidesequence identity to SEQ. ID. Nos. 1-26 or nucleic acid residues601-1000 of SEQ ID NO: 26 namely that exhibits at least 80% sequenceidentity, at least 85%, at least 90%, and at least 95%, 96%, 97%, 98% or99% sequence identity compared to SEQ. ID. Nos. 1-26 or residues601-1000 of SEQ ID NO: 26. Such sequence identity can be calculated bythe algorithms and computers programs described above.

The present invention further encompasses “functional variants” or“function fragments” of the disclosed sequences, particularly fragmentsof SEQ ID NOs: 1-26 and residues 601-1000 of SEQ ID NO: 5 that retainpromoter activity. Functional variants include, for example, regulatorysequences of the invention having one or more nucleotide substitutions,deletions or insertions and wherein the variant retains promoteractivity. Functional variants can be created by any of a number ofmethods available to one skilled in the art, such as by site-directedmutagenesis, induced mutation, identified as allelic variants, cleavingthrough use of restriction enzymes, or the like. Activity can likewisebe measured by any variety of techniques, including measurement ofreporter activity as is described at U.S. Pat. No. 6,844,484, Northernblot analysis, or similar techniques. The '484 patent describes theidentification of functional variants of different promoters.

Functional fragment, that is, a regulatory sequence fragment can beformed by one or more deletions from a larger regulatory element. Forexample, in some instances, the 5′ portion of a promoter up to the TATAbox near the transcription start site can be deleted without abolishingpromoter activity, as described by Opsahl-Sorteberg, H-G. et al.,“Identification of a 49-bp fragment of the HvLTP2 promoter directingaleruone cell specific expression” Gene 341:49-58 (2004). Such fragmentsshould retain promoter activity, particularly the ability to driveexpression of operably linked nucleotide sequences. Activity can bemeasured by Northern blot analysis, reporter activity measurements whenusing transcriptional fusions, and the like. See, for example, Sambrooket al., Molecular Cloning, A laboratory Manual (1989). Functionalfragments can be obtained by use of restriction enzymes to cleave thenaturally occurring regulatory element nucleotide sequences disclosedherein; by synthesizing a nucleotide sequence from the naturallyoccurring DNA sequence; or can be obtained through the use of PCRtechnology. See particularly, Mullis et al., Methods Enzymol.,155:335-350 (1987) and Erlich, ed., PCR Technology (Stockton Press, NewYork), (1989).

For example, a routine way to remove part of a DNA sequence is to use anexonuclease in combination with DNA amplification to produceunidirectional nested deletions of double stranded DNA clones. Acommercial kit for this purpose is sold under the trade name Exo-Size™(New England Biolabs, Beverly, Mass.). Briefly, this procedure entailsincubating exonuclease III with DNA to progressively remove nucleotidesin the 3′ to 5′ direction at 5′ overhangs, blunt ends or nicks in theDNA template. However, exonuclease III is unable to remove nucleotidesat 3′, 4-base overhangs. Timed digests of a clone with this enzymeproduces unidirectional nested deletions.

5. TESTING OF PROMOTERS

Promoters of the invention, including functional fragments, are testedfor activity by cloning the sequence into an appropriate vector,transforming plants with the construct and assaying for marker geneexpression. Recombinant DNA constructs are prepared which comprise thepromoter sequences of the invention inserted into a vector suitable fortransformation of plant cells. The construct can be made using standardrecombinant DNA techniques (Sambrook et al. 1989) and can be introducedto the species of interest by Agrobacterium-mediated transformation orby other means of transformation as referenced below.

The vector backbone can be any of those typical in the art such asplasmids, viruses, artificial chromosomes, BACs, YACs and PACs andvectors of the sort described by

-   (a) BAC: Shizuya et al. (1992) Proc. Natl. Acad. Sci. USA 89:    8794-8797; Hamilton et al. (1996) Proc. Natl. Acad. Sci. USA 93:    9975-9979;-   (b) YAC: Burke et al. (1987) Science 236:806-812;-   (c) PAC: Sternberg N. et al. (1990) Proc Natl Acad Sci USA.    87(1):103-7;-   (d) Bacteria-Yeast Shuttle Vectors: Bradshaw et al. (1995) Nucl    Acids Res 23: 4850-4856;-   (e) Lambda Phage Vectors: Replacement Vector, e.g., Frischauf et    al. (1983) J. Mol Biol 170: 827-842; or Insertion vector, e.g.,    Huynh et al. (1985) In: Glover N M (ed) DNA Cloning: A practical    Approach, Vol. 1 Oxford: IRL Press; T-DNA gene fusion vectors:    Walden et al. (1990) Mol Cell Biol 1: 175-194; and-   (g) Plasmid vectors: Sambrook et al., infra.

Typically, the construct comprises a vector containing a promotersequence of the present invention operationally linked to any markergene. The promoter was identified as a promoter by the expression of themarker gene. Although many marker genes can be used, Green FluorescentProtein (GFP) is preferred. The vector may also comprise a marker genethat confers a selectable phenotype on plant cells. The marker mayencode biocide resistance, particularly antibiotic resistance, such asresistance to kanamycin, G418, bleomycin, hygromycin, or herbicideresistance, such as resistance to chlorosulfuron or phosphinotricin.Vectors can also include origins of replication, scaffold attachmentregions (SARs), markers, homologous sequences, introns, etc.

6. CONSTRUCTING PROMOTERS WITH CONTROL ELEMENTS

6.1 Combining Promoters and Promoter Control Elements

The promoter and promoter control elements of the present invention,both naturally occurring and synthetic, can be used alone or combinedwith each other to produce the desired preferential transcription. Also,the promoters of the invention can be combined with other knownsequences to obtain other useful promoters to modulate, for example,tissue transcription specific or transcription specific to certainconditions. Such preferential transcription can be determined using thetechniques or assays described above.

Promoters can contain any number of control elements. For example, apromoter can contain multiple transcription binding sites or othercontrol elements. One element may confer tissue or organ specificity;another element may limit transcription to specific time periods, etc.Typically, promoters will contain at least a basal or core promoter asdescribed above. Any additional element can be included as desired. Forexample, a fragment comprising a basal or “core” promoter can be fusedwith another fragment with any number of additional control elements.

The following are promoters that are induced under stress conditions andcan be combined with those of the present invention: ldh1 (oxygenstress; tomato; see Germain and Ricard (1997) Plant Mol Biol 35:949-54),GPx and CAT (oxygen stress; mouse; see Franco et al. (1999) Free RadicBiol Med 27:1122-32), ci7 (cold stress; potato; see Kirch et al. (1997)Plant Mol Biol. 33:897-909), Bz2 (heavy metals; maize; see Marrs andWalbot (1997) Plant Physiol 113:93-102), HSP32 (hyperthermia; rat; seeRaju and Maines (1994) Biochim Biophys Acta 1217:273-80), and MAPKAPK-2(heat shock; Drosophila; see Larochelle and Suter (1995) Gene163:209-14).

In addition, the following examples of promoters are induced by thepresence or absence of light can be used in combination with those ofthe present invention: Topoisomerase II (pea; see Reddy et al. (1999)Plant Mol Biol 41:125-37), chalcone synthase (soybean; see Wingender etal. (1989) Mol Gen Genet 218:315-22) mdm2 gene (human tumor; see Saucedoet al. (1998) Cell Growth Differ 9:119-30), Clock and BMAL1 (rat; seeNamihira et al. (1999) Neurosci Lett 271:1-4, PHYA (Arabidopsis; seeCanton and Quail (1999) Plant Physiol 121:1207-16), PRB-1b (tobacco; seeSessa et al. (1995) Plant Mol Biol 28:537-47) and Ypr10 (common bean;see Walter et al. (1996) Eur J Biochem 239:281-93).

The promoters and control elements of the following genes can be used incombination with the present invention to confer tissue specificity:MipB (iceplant; Yamada et al. (1995) Plant Cell 7:1129-42) and SUCS(root nodules; broadbean; Kuster et al. (1993) Mol Plant MicrobeInteract 6:507-14) for roots, OsSUT1 (rice; Hirose et al. (1997) PlantCell Physiol 38:1389-96) for leaves, Msg (soybean; Stomvik et al. (1999)Plant Mol Biol 41:217-31) for siliques, cell (Arabidopsis; Shani et al.(1997) Plant Mol Biol 34(6):837-42) and ACT11 (Arabidopsis; Huang et al.(1997) Plant Mol Biol 33:125-39) for inflorescence.

Still other promoters are affected by hormones or participate inspecific physiological processes, which can be used in combination withthose of present invention. Some examples are the ACC synthase gene thatis induced differently by ethylene and brassinosteroids (mung bean; Yiet al. (1999) Plant Mol Biol 41:443-54), the TAPG1 gene that is activeduring abscission (tomato; Kalaitzis et al. (1995) Plant Mol Biol28:647-56), and the 1-aminocyclopropane-1-carboxylate synthase gene(carnation; Jones et al. (1995) Plant Mol Biol 28:505-12) and theCP-2/cathepsin L gene (rat; Kim and Wright (1997) Biol Reprod57:1467-77), both active during senescence.

Spacing between control elements or the configuration or controlelements can be determined or optimized to permit the desiredprotein-polynucleotide or polynucleotide interactions to occur.

For example, if two transcription factors bind to a promotersimultaneously or relatively close in time, the binding sites are spacedto allow each factor to bind without steric hindrance. The spacingbetween two such hybridizing control elements can be as small as aprofile of a protein bound to a control element. In some cases, twoprotein binding sites can be adjacent to each other when the proteinsbind at different times during the transcription process.

Further, when two control elements hybridize the spacing between suchelements will be sufficient to allow the promoter polynucleotide tohairpin or loop to permit the two elements to bind. The spacing betweentwo such hybridizing control elements can be as small as a t-RNA loop,to as large as 10 kb.

Typically, the spacing is no smaller than 5 bases; more typically, nosmaller than 8; more typically, no smaller than 15 bases; moretypically, no smaller than 20 bases; more typically, no smaller than 25bases; even more typically, no smaller than 30, 35, 40 or 50 bases.

Usually, the fragment size in no larger than 5 kb bases; more usually,no larger than 2 kb; more usually, no larger than 1 kb; more usually, nolarger than 800 bases; more usually, no larger than 500 bases; even moreusually, no more than 250, 200, 150 or 100 bases. In some embodiments,the nucleic acid of the invention comprises at least one fragment ofYP0286 (SEQ ID NO:5), e.g., YP2219 (SEQ ID NO:4), with the proviso thatsaid nucleic acid does not consist of YP0286 (SEQ ID NO:5).

Such spacing between promoter control elements can be determined usingthe techniques and assays described above.

6.2 Vectors Used to Transform Cells/Hosts

A plant transformation construct containing a promoter of the presentinvention may be introduced into plants by any plant transformationmethod. Methods and materials for transforming plants by introducing aplant expression construct into a plant genome in the practice of thisinvention can include any of the well-known and demonstrated methodsincluding electroporation (U.S. Pat. No. 5,384,253); microprojectilebombardment (U.S. Pat. No. 5,015,580; U.S. Pat. No. 5,550,318; U.S. Pat.No. 5,538,880; U.S. Pat. No. 6,160,208; U.S. Pat. No. 6,399,861; andU.S. Pat. No. 6,403,865); Agrobacterium-mediated transformation (U.S.Pat. No. 5,824,877; U.S. Pat. No. 5,591,616; U.S. Pat. No. 5,981,840;and U.S. Pat. No. 6,384,301); and protoplast transformation (U.S. Pat.No. 5,508,184).

The present promoters and/or promoter control elements may be deliveredto a system such as a cell by way of a vector. For the purposes of thisinvention, such delivery may range from simply introducing the promoteror promoter control element by itself randomly into a cell tointegration of a cloning vector containing the present promoter orpromoter control element. Thus, a vector need not be limited to a DNAmolecule such as a plasmid, cosmid or bacterial phage that has thecapability of replicating autonomously in a host cell. All other mannerof delivery of the promoters and promoter control elements of theinvention are envisioned. The various T-DNA vector types are a preferredvector for use with the present invention. Many useful vectors arecommercially available.

It may also be useful to attach a marker sequence to the presentpromoter and promoter control element in order to determine activity ofsuch sequences. Marker sequences typically include genes that provideantibiotic resistance, such as tetracycline resistance, hygromycinresistance or ampicillin resistance, or provide herbicide resistance.Specific selectable marker genes may be used to confer resistance toherbicides such as glyphosate, glufosinate or broxynil (Comai et al.(1985) Nature 317: 741-744; Gordon-Kamm et al. (1990) Plant Cell 2:603-618; and Stalker et al. (1988) Science 242: 419-423). Other markergenes exist which provide hormone responsiveness.

The promoter or promoter control element of the present invention may beoperably linked to a polynucleotide to be transcribed. In this manner,the promoter or promoter control element may modify transcription bymodulating transcript levels of that polynucleotide when inserted into agenome.

However, prior to insertion into a genome, the promoter or promotercontrol element need not be linked, operably or otherwise, to apolynucleotide to be transcribed. For example, the promoter or promotercontrol element may be inserted alone into the genome in front of apolynucleotide already present in the genome. In this manner, thepromoter or promoter control element may modulate the transcription of apolynucleotide that was already present in the genome. Thispolynucleotide may be native to the genome or inserted at an earliertime.

Alternatively, the promoter or promoter control element may be insertedinto a genome alone to modulate transcription. See, for example,Vaucheret, H et al. (1998) Plant J 16: 651-659. Rather, the promoter orpromoter control element may be simply inserted into a genome ormaintained extrachromosomally as a way to divert transcription resourcesof the system to itself. This approach may be used to downregulate thetranscript levels of a group of polynucleotide(s).

The nature of the polynucleotide to be transcribed is not limited.Specifically, the polynucleotide may include sequences that will haveactivity as RNA as well as sequences that result in a polypeptideproduct. These sequences may include, but are not limited to antisensesequences, RNAi sequences, ribozyme sequences, spliceosomes, amino acidcoding sequences, and fragments thereof. Specific coding sequences mayinclude, but are not limited to endogenous proteins or fragmentsthereof, or heterologous proteins including marker genes or fragmentsthereof.

Constructs of the present invention would typically contain a promoteroperably linked to a transcribable nucleic acid molecule operably linkedto a 3′ transcription termination nucleic acid molecule. In addition,constructs may include but are not limited to additional regulatorynucleic acid molecules from the 3′-untranslated region (3′ UTR) of plantgenes (e.g., a 3′ UTR to increase mRNA stability of the mRNA, such asthe PI-II termination region of potato or the octopine or nopalinesynthase 3′ termination regions). Constructs may include but are notlimited to the 5′ untranslated regions (5′ UTR) of an mRNA nucleic acidmolecule which can play an important role in translation initiation andcan also be a genetic component in a plant expression construct. Forexample, non-translated 5′ leader nucleic acid molecules derived fromheat shock protein genes have been demonstrated to enhance geneexpression in plants (see for example, U.S. Pat. No. 5,659,122 and U.S.Pat. No. 5,362,865, all of which are hereby incorporated by reference).These additional upstream and downstream regulatory nucleic acidmolecules may be derived from a source that is native or heterologouswith respect to the other elements present on the promoter construct.

Thus, one embodiment of the invention is a promoter such as provided inSEQ ID NOs: 1-26 or residues 601-1000 of SEQ ID NO: 26, operably linkedto a transcribable nucleic acid molecule so as to direct transcriptionof said transcribable nucleic acid molecule at a desired level or in adesired tissue or developmental pattern upon introduction of saidconstruct into a plant cell. In some cases, the transcribable nucleicacid molecule comprises a protein-coding region of a gene, and thepromoter provides for transcription of a functional mRNA molecule thatis translated and expressed as a protein product. Constructs may also beconstructed for transcription of antisense RNA molecules or othersimilar inhibitory RNA in order to inhibit expression of a specific RNAmolecule of interest in a target host cell.

Exemplary transcribable nucleic acid molecules for incorporation intoconstructs of the present invention include, for example, nucleic acidmolecules or genes from a species other than the target gene species, oreven genes that originate with or are present in the same species, butare incorporated into recipient cells by genetic engineering methodsrather than classical reproduction or breeding techniques. Exogenousgene or genetic element is intended to refer to any gene or nucleic acidmolecule that is introduced into a recipient cell. The type of nucleicacid molecule included in the exogenous nucleic acid molecule caninclude a nucleic acid molecule that is already present in the plantcell, a nucleic acid molecule from another plant, a nucleic acidmolecule from a different organism, or a nucleic acid molecule generatedexternally, such as a nucleic acid molecule containing an antisensemessage of a gene, or a nucleic acid molecule encoding an artificial ormodified version of a gene.

The promoters of the present invention can be incorporated into aconstruct using marker genes as described, and tested in transientanalyses that provide an indication of gene expression in stable plantsystems. As used herein the term “marker gene” refers to anytranscribable nucleic acid molecule whose expression can be screened foror scored in some way. Methods of testing for marker gene expression intransient assays are known to those of skill in the art. Transientexpression of marker genes has been reported using a variety of plants,tissues, plant cell(s), and DNA delivery systems. For example, types oftransient analyses can include but are not limited to direct genedelivery via electroporation or particle bombardment of tissues in anytransient plant assay using any plant species of interest. Suchtransient systems would include, but are not limited to, electroporationof protoplasts from a variety of tissue sources or particle bombardmentof specific tissues of interest. The present invention encompasses theuse of any transient expression system to evaluate promoters or promoterfragments operably linked to any transcribable nucleic acid molecules,including but not limited to selected reporter genes, marker genes, orgenes of agronomic interest. Examples of plant tissues envisioned totest in transients via an appropriate delivery system would include, butare not limited to, leaf base tissues, callus, cotyledons, roots,endosperm, embryos, floral tissue, pollen, and epidermal tissue.

Promoters and control elements of the present invention are useful formodulating metabolic or catabolic processes. Such processes include, butare not limited to, secondary product metabolism, amino acid synthesis,seed protein storage, increased biomass, oil development, pest defenseand nitrogen usage. Some examples of genes, transcripts and peptides orpolypeptides participating in these processes, which can be modulated bythe present invention: are tryptophan decarboxylase (tdc) andstrictosidine synthase (str1), dihydrodipicolinate synthase (DHDPS) andaspartate kinase (AK), 2S albumin and alpha-, beta-, and gamma-zeins,ricinoleate and 3-ketoacyl-ACP synthase (KAS), Bacillus thuringiensis(Bt) insecticidal protein, cowpea trypsin inhibitor (CpTI), asparaginesynthetase and nitrite reductase. Alternatively, expression constructscan be used to inhibit expression of these peptides and polypeptides byincorporating the promoters in constructs for antisense use,co-suppression use or for the production of dominant negative mutations.

As explained above, several types of regulatory elements existconcerning transcription regulation. Each of these regulatory elementsmay be combined with the present vector if desired. Translation ofeukaryotic mRNA is often initiated at the codon that encodes the firstmethionine. Thus, when constructing a recombinant polynucleotideaccording to the present invention for expressing a protein product, itis preferable to ensure that the linkage between the 3′ portion,preferably including the TATA box, of the promoter and thepolynucleotide to be transcribed, or a functional derivative thereof,does not contain any intervening codons which are capable of encoding amethionine.

The vector of the present invention may contain additional components.For example, an origin of replication allows for replication of thevector in a host cell. Additionally, homologous sequences flanking aspecific sequence allow for specific recombination of the specificsequence at a desired location in the target genome. T-DNA sequencesalso allow for insertion of a specific sequence randomly into a targetgenome.

The vector may also be provided with a plurality of restriction sitesfor insertion of a polynucleotide to be transcribed as well as thepromoter and/or promoter control elements of the present invention. Thevector may additionally contain selectable marker genes. The vector mayalso contain a transcriptional and translational initiation region, anda transcriptional and translational termination region functional in thehost cell. The termination region may be native with the transcriptionalinitiation region, may be native with the polynucleotide to betranscribed, or may be derived from another source. Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.See also, Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144;Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev.5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al.(1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res.17:7891-7903; Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

Where appropriate, the polynucleotide to be transcribed may be optimizedfor increased expression in a certain host cell. For example, thepolynucleotide can be synthesized using preferred codons for improvedtranscription and translation. See U.S. Pat. Nos. 5,380,831, 5,436,391;see also and Murray et al. (1989) Nucleic Acids Res. 17:477-498.

Additional sequence modifications include elimination of sequencesencoding spurious polyadenylation signals, exon intron splice sitesignals, transposon-like repeats, and other such sequences wellcharacterized as deleterious to expression. The G-C content of thepolynucleotide may be adjusted to levels average for a given cellularhost, as calculated by reference to known genes expressed in the hostcell. The polynucleotide sequence may be modified to avoid hairpinsecondary mRNA structures.

A general description of expression vectors and reporter genes can befound in Umber, et al. (1993) “Vectors for Plant Transformation” InMethods in Plant Molecular Biology & Biotechnology, Glich et al. Eds.pp. 89-119, CRC Press. Moreover GUS expression vectors and GUS genecassettes are available from Clonetech Laboratories, Inc., Palo Alto,Calif. while luciferase expression vectors and luciferase gene cassettesare available from Promega Corp. (Madison, Wis.). GFP vectors areavailable from Aurora Biosciences.

6.3 Polynucleotide Insertion into a Host Cell

The promoters according to the present invention can be inserted into ahost cell. A host cell includes but is not limited to a plant,mammalian, insect, yeast, and prokaryotic cell, preferably a plant cell.

The method of insertion into the host cell genome is chosen based onconvenience. For example, the insertion into the host cell genome mayeither be accomplished by vectors that integrate into the host cellgenome or by vectors which exist independent of the host cell genome.

The promoters of the present invention can exist autonomously orindependent of the host cell genome. Vectors of these types are known inthe art and include, for example, certain type of non-integrating viralvectors, autonomously replicating plasmids, artificial chromosomes, andthe like.

Additionally, in some cases transient expression of a promoter may bedesired.

The promoter sequences, promoter control elements or vectors of thepresent invention may be transformed into host cells. Thesetransformations may be into protoplasts or intact tissues or isolatedcells. Preferably expression vectors are introduced into intact tissue.General methods of culturing plant tissues are provided for example byMaki et al. (1993) “Procedures for Introducing Foreign DNA into Plants”In Methods in Plant Molecular Biology & Biotechnology, Glich et al. Eds.pp. 67-88 CRC Press; and by Phillips et al. (1988) “Cell-Tissue Cultureand In-Vitro Manipulation” In Corn & Corn Improvement, 3rd EditionSprague et al. eds., pp. 345-387, American Society of Agronomy Inc. etal.

Methods of introducing polynucleotides into plant tissue include thedirect infection or co-cultivation of plant cell with Agrobacteriumtumefaciens, Horsch et al. (1985) Science, 227:1229. Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer provided by Gruber et al. supra.

Alternatively, polynucleotides are introduced into plant cells or otherplant tissues using a direct gene transfer method such asmicroprojectile-mediated delivery, DNA injection, electroporation andthe like. More preferably polynucleotides are introduced into planttissues using the microprojectile media delivery with the biolisticdevice. See, for example, Tomes et al., “Direct DNA transfer into intactplant cells via microprojectile bombardment” In: Gamborg and Phillips(Eds.) Plant Cell, Tissue and Organ Culture: Fundamental Methods,Springer Verlag, Berlin (1995).

Methods for specifically transforming dicots are well known to thoseskilled in the art. Transformation and plant regeneration using thesemethods have been described for a number of crops including, but notlimited to, cotton (Gossypium hirsutum), soybean (Glycine max), peanut(Arachis hypogaea), and members of the genus Brassica.

Methods for transforming monocots are well known to those skilled in theart. Transformation and plant regeneration using these methods have beendescribed for a number of crops including, but not limited to, barley(Hordeum vulgarae); maize (Zea mays); oats (Avena sativa); orchard grass(Dactylis glomerata); rice (Oryza sativa, including indica and japonicavarieties); sorghum (Sorghum bicolor); sugar cane (Saccharum sp); tallfescue (Festuca arundinacea); turfgrass species (e.g. species: Agrostisstolonifera, Poa pratensis, Stenotaphrum secundatum); wheat (Triticumaestivum), switchgrass (Panicum vigatum) and alfalfa (Medicago sativa).It is apparent to those of skill in the art that a number oftransformation methodologies can be used and modified for production ofstable transgenic plants from any number of target plants of interest.

The polynucleotides and vectors described herein can be used totransform a number of monocotyledonous and dicotyledonous plants andplant cell systems, including species from one of the followingfamilies: Acanthaceae, Alliaceae, Alstroemeriaceae, Amaryllidaceae,Apocynaceae, Arecaceae, Asteraceae, Berberidaceae, Bixaceae,Brassicaceae, Bromeliaceae, Cannabaceae, Caryophyllaceae,Cephalotaxaceae, Chenopodiaceae, Colchicaceae, Cucurbitaceae,Dioscoreaceae, Ephedraceae, Erythroxylaceae, Euphorbiaceae, Fabaceae,Lamiaceae, Linaceae, Lycopodiaceae, Malvaceae, Melanthiaceae, Musaceae,Myrtaceae, Nyssaceae, Papaveraceae, Pinaceae, Plantaginaceae, Poaceae,Rosaceae, Rubiaceae, Salicaceae, Sapindaceae, Solanaceae, Taxaceae,Theaceae, or Vitaceae.

Suitable species may include members of the genus Abelmoschus, Abies,Acer, Agrostis, Allium, Alstroemeria, Ananas, Andrographis, Andropogon,Artemisia, Arundo, Atropa, Berberis, Beta, Bixa, Brassica, Calendula,Camellia, Camptotheca, Cannabis, Capsicum, Carthamus, Catharanthus,Cephalotaxus, Chrysanthemum, Cinchona, Citrullus, Coffea, Colchicum,Coleus, Cucumis, Cucurbita, Cynodon, Datura, Dianthus, Digitalis,Dioscorea, Elaeis, Ephedra, Erianthus, Erythroxylum, Eucalyptus,Festuca, Fragaria, Galanthus, Glycine, Gossypium, Helianthus, Hevea,Hordeum, Hyoscyamus, Jatropha, Lactuca, Linum, Lolium, Lupinus,Lycopersicon, Lycopodium, Manihot, Medicago, Mentha, Miscanthus, Musa,Nicotiana, Oryza, Panicum, Papaver, Parthenium, Pennisetum, Petunia,Phalaris, Phleum, Pinus, Poa, Poinsettia, Populus, Rauwolfia, Ricinus,Rosa, Saccharum, Salix, Sanguinaria, Scopolia, Secale, Solanum, Sorghum,Spartina, Spinacea, Tanacetum, Taxus, Theobroma, Triticosecale,Triticum, Uniola, Veratrum, Vinca, Vitis, and Zea.

Suitable species include Panicum spp. or hybrids thereof, Sorghum spp.or hybrids thereof, sudangrass, Miscanthus spp. or hybrids thereof,Saccharum spp. or hybrids thereof, Erianthus spp., Populus spp.,Andropogon gerardii (big bluestem), Pennisetum purpureum (elephantgrass) or hybrids thereof (e.g., Pennisetum purpureum×Pennisetumtyphoidum), Phalaris arundinacea (reed canarygrass), Cynodon dactylon(bermudagrass), Festuca arundinacea (tall fescue), Spartina pectinata(prairie cord-grass), Medicago sativa (alfalfa), Arundo donax (giantreed) or hybrids thereof, Secale cereale (rye), Salix spp. (willow),Eucalyptus spp. (eucalyptus), Triticosecale (Triticum—wheat X rye),Tripsicum dactyloides (Eastern gammagrass), Leymus cinereus (basinwildrye), Leymus condensatus (giant wildrye), and bamboo.

In some embodiments, a suitable species can be a wild, weedy, orcultivated sorghum species such as, but not limited to, Sorghum almum,Sorghum amplum, Sorghum angustum, Sorghum arundinaceum, Sorghum bicolor(such as bicolor, guinea, caudatum, kafir, and durra), Sorghumbrachypodum, Sorghum bulbosum, Sorghum burmahicum, Sorghum controversum,Sorghum drummondii, Sorghum ecarinatum, Sorghum exstans, Sorghum grande,Sorghum halepense, Sorghum interjectum, Sorghum intrans, Sorghumlaxifiorum, Sorghum leiocladum, Sorghum macrospermum, Sorghummatarankense, Sorghum miliaceum, Sorghum nigrum, Sorghum nitidum,Sorghum plumosum, Sorghum propinquum, Sorghum purpureosericeum, Sorghumstipoideum, Sorghum sudanensese, Sorghum timorense, Sorghumtrichocladum, Sorghum versicolor, Sorghum virgatum, Sorghum vulgare, orhybrids such as Sorghum×almum, Sorghum×sudangrass or Sorghum×drummondii.

Suitable species also include Helianthus annuus (sunflower), Carthamustinctorius (safflower), Jatropha curcas (Jatropha), Ricinus communis(castor), Elaeis guineensis (palm), Linum usitatissimum (flax), andBrassica juncea.

Suitable species also include Beta vulgaris (sugarbeet), and Manihotesculenta (cassava).

Suitable species also include Lycopersicon esculentum (tomato), Lactucasativa (lettuce), Musa paradisiaca (banana), Solanum tuberosum (potato),Brassica oleracea (broccoli, cauliflower, brusselsprouts), Camelliasinensis (tea), Fragaria ananassa (strawberry), Theobroma cacao (cocoa),Coffea arabica (coffee), Vitis vinifera (grape), Ananas comosus(pineapple), Capsicum annum (hot & sweet pepper), Allium cepa (onion),Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima(squash), Cucurbita moschata (squash), Spinacea oleracea (spinach),Citrullus lanatus (watermelon), Abelmoschus esculentus (okra), andSolanum melongena (eggplant).

Suitable species also include Papaver somniferum (opium poppy), Papaverorientale, Taxus baccata, Taxus brevifolia, Artemisia annua, Cannabissativa, Camptotheca acuminate, Catharanthus roseus, Vinca rosea,Cinchona officinalis, Colchicum autumnale, Veratrum californica,Digitalis lanata, Digitalis purpurea, Dioscorea spp., Andrographispaniculata, Atropa belladonna, Datura stomonium, Berberis spp.,Cephalotaxus spp., Ephedra sinica, Ephedra spp., Erythroxylum coca,Galanthus wornorii, Scopolia spp., Lycopodium serratum (=Huperziaserrata), Lycopodium spp., Rauwolfia serpentina, Rauwolfia spp.,Sanguinaria canadensis, Hyoscyamus spp., Calendula officinalis,Chrysanthemum parthenium, Coleus forskohlii, and Tanacetum parthenium.

Suitable species also include Parthenium argentatum (guayule), Heveaspp. (rubber), Mentha spicata (mint), Mentha piperita (mint), Bixaorellana, and Alstroemeria spp.

Suitable species also include Rosa spp. (rose), Dianthus caryophyllus(carnation), Petunia spp. (petunia) and Poinsettia pulcherrima(poinsettia).

Suitable species also include Nicotiana tabacum (tobacco), Lupinus albus(lupin), Uniola paniculata (oats), bentgrass (Agrostis spp.), Populustremuloides (aspen), Pinus spp. (pine), Abies spp. (fir), Acer spp.(maple, Hordeum vulgare (barley), Poa pratensis (bluegrass), Lolium spp.(ryegrass) and Phleum pratense (timothy).

Thus, the methods and compositions can be used over a broad range ofplant species, including species from the dicot genera Brassica,Carthamus, Glycine, Gossypium, Helianthus, Jatropha, Parthenium,Populus, and Ricinus; and the monocot genera Elaeis, Festuca, Hordeum,Lolium, Oryza, Panicum, Pennisetum, Phleum, Poa, Saccharum, Secale,Sorghum, Triticosecale, Triticum, and Zea. In some embodiments, a plantis a member of the species Panicum virgatum (switchgrass), Sorghumbicolor (sorghum, sudangrass), Miscanthus giganteus (miscanthus),Saccharum sp. (energycane), Populus balsamifera (poplar), Zea mays(corn), Glycine max (soybean), Brassica napus (canola), Triticumaestivum (wheat), Gossypium hirsutum (cotton), Oryza sativa (rice),Helianthus annuus (sunflower), Medicago sativa (alfalfa), Beta vulgaris(sugarbeet), or Pennisetum glaucum (pearl millet).

In certain embodiments, the polynucleotides and vectors described hereincan be used to transform a number of monocotyledonous and dicotyledonousplants and plant cell systems, wherein such plants are hybrids ofdifferent species or varieties of a specific species (e.g., Saccharumsp.×Miscanthus sp., Panicum virgatum×Panicum amarum, Panicumvirgatum×Panicum amarulum, and Pennisetum purpureum×Pennisetumtyphoidum).

In another embodiment of the current invention, expression constructscan be used for gene expression in callus culture for the purpose ofexpressing marker genes encoding peptides or polypeptides that allowidentification of transformed plants. Here, a promoter that isoperatively linked to a polynucleotide to be transcribed is transformedinto plant cells and the transformed tissue is then placed oncallus-inducing media. If the transformation is conducted with leafdiscs, for example, callus will initiate along the cut edges. Oncecallus growth has initiated, callus cells can be transferred to callusshoot-inducing or callus root-inducing media. Gene expression will occurin the callus cells developing on the appropriate media: callusroot-inducing promoters will be activated on callus root-inducing media,etc. Examples of such peptides or polypeptides useful as transformationmarkers include, but are not limited to barstar, glyphosate,chloramphenicol acetyltransferase (CAT), kanamycin, spectinomycin,streptomycin or other antibiotic resistance enzymes, green fluorescentprotein (GFP), and β-glucuronidase (GUS), etc. Some of the promotersprovided in SEQ ID NOs: 1-26 or nucleic acid residues 601-1000 of SEQ IDNO: 26 will also be capable of sustaining expression in some tissues ororgans after the initiation or completion of regeneration. Examples ofthese tissues or organs are somatic embryos, cotyledon, hypocotyl,epicotyl, leaf, stems, roots, flowers and seed.

Integration into the host cell genome also can be accomplished bymethods known in the art, for example, by the homologous sequences orT-DNA discussed above or using the cre-lox system (A. C. Vergunst et al.(1998) Plant Mol. Biol. 38:393).

7. USES OF THE PROMOTERS OF THE INVENTION

7.1 Use of the Promoters to Study and Screen for Expression

The promoters of the present invention can be used to further understanddevelopmental mechanisms. For example, promoters that are specificallyinduced during callus formation, somatic embryo formation, shootformation or root formation can be used to explore the effects ofoverexpression, repression or ectopic expression of target genes, or forisolation of trans-acting factors.

The vectors of the invention can be used not only for expression ofcoding regions but may also be used in exon-trap cloning, or promotertrap procedures to detect differential gene expression in varioustissues (see Lindsey et al. (1993) Transgenic Research 2:3347. Auch andReth (1990) Nucleic Acids Research 18: 6743).

Entrapment vectors, first described for use in bacteria (Casadaban andCohen (1979) Proc. Nat. Aca. Sci. U.S.A. 76: 4530; Casadaban et al.(1980) J. Bacteriol. 143: 971) permit selection of insertional eventsthat lie within coding sequences. Entrapment vectors can be introducedinto pluripotent ES cells in culture and then passed into the germlinevia chimeras (Gossler et al. aaa91989) Science 244: 463; Skarnes (1990)Biotechnology 8: 827). Promoter or gene trap vectors often contain areporter gene, e.g., lacZ, lacking its own promoter and/or spliceacceptor sequence upstream. That is, promoter gene traps contain areporter gene with a splice site but no promoter. If the vector lands ina gene and is spliced into the gene product, then the reporter gene isexpressed.

Recently, the isolation of preferentially-induced genes has been madepossible with the use of sophisticated promoter traps (e.g. IVET) thatare based on conditional auxotrophy complementation or drug resistance.In one IVET approach, various bacterial genome fragments are placed infront of a necessary metabolic gene coupled to a reporter gene. The DNAconstructs are inserted into a bacterial strain otherwise lacking themetabolic gene, and the resulting bacteria are used to infect the hostorganism. Only bacteria expressing the metabolic gene survive in thehost organism; consequently, inactive constructs can be eliminated byharvesting only bacteria that survive for some minimum period in thehost. At the same time, broadly active constructs can be eliminated byscreening only bacteria that do not express the reporter gene underlaboratory conditions. The bacteria selected by such a method containconstructs that are selectively induced only during infection of thehost. The IVET approach can be modified for use in plants to identifygenes induced in either the bacteria or the plant cells upon pathogeninfection or root colonization. For information on IVET see the articlesby Mahan et al. (1993) Science 259:686-688, Mahan et al. (1995) Proc.Natl. Acad. Sci. USA 92:669-673, Heithoff et al. (1997) Proc. Natl.Acad. Sci USA 94:934-939, and Wang et al. (1996) Proc. Natl. Acad. SciUSA 93:10434.

7.2 Use of the Promoters to Transcribe Genes of Interest

In one embodiment of the invention, a nucleic acid molecule as shown inSEQ ID NOs: 1-26 or nucleic acid residues 601-1000 of SEQ ID NO: 26 isincorporated into a construct such that a promoter of the presentinvention is operably linked to a transcribable nucleic acid moleculethat is a gene of agronomic interest. As used herein, the term “gene ofagronomic interest” refers to a transcribable nucleic acid molecule thatincludes but is not limited to a gene that provides a desirablecharacteristic associated with plant morphology, physiology, growth anddevelopment, yield, nutritional enhancement, disease or pest resistance,or environmental or chemical tolerance. The expression of a gene ofagronomic interest is desirable in order to confer an agronomicallyimportant trait. A gene of agronomic interest that provides a beneficialagronomic trait to crop plants may be, for example, including, but notlimited to genetic elements comprising herbicide resistance, increasedyield, increased biomass, insect control, fungal disease resistance,virus resistance, nematode resistance, bacterial disease resistance,starch production, modified oils production, high oil production,modified fatty acid content, high protein production, fruit ripening,enhanced animal and human nutrition, biopolymers, environmental stressresistance, pharmaceutical peptides, improved processing traits,improved digestibility, industrial enzyme production, improved flavor,nitrogen fixation, hybrid seed production, and biofuel production. Thegenetic elements, methods, and transgenes described in the patentslisted above are hereby incorporated by reference.

Alternatively, a transcribable nucleic acid molecule can effect theabove mentioned phenotypes by encoding a RNA molecule that causes thetargeted inhibition of expression of an endogenous gene, for example viaantisense, inhibitory RNA (RNAi), or cosuppression-mediated mechanisms.The RNA could also be a catalytic RNA molecule (i.e., a ribozyme)engineered to cleave a desired endogenous mRNA product. Thus, anynucleic acid molecule that encodes a protein or mRNA that expresses aphenotype or morphology change of interest may be useful for thepractice of the present invention.

7.3. Stress Induced Preferential Transcription

Promoters and control elements providing modulation of transcriptionunder oxidative, drought, oxygen, wound, and methyl jasmonate stress areparticularly useful for producing host cells or organisms that are moreresistant to biotic and abiotic stresses. In a plant, for example,modulation of genes, transcripts, and/or polypeptides in response tooxidative stress can protect cells against damage caused by oxidativeagents, such as hydrogen peroxide and other free radicals.

Drought induction of genes, transcripts, and/or polypeptides are usefulto increase the viability of a plant, for example, when water is alimiting factor. In contrast, genes, transcripts, and/or polypeptidesinduced during oxygen stress can help the flood tolerance of a plant.

The promoters and control elements of the present invention can modulatestresses similar to those described in, for example, stress conditionsare VuPLD1 (drought stress; Cowpea; see Pham-Thi et al. (1999) Plant MolBiol 39:1257-65), pyruvate decarboxylase (oxygen stress; rice; seeRivosal et al. (1997) Plant Physiol 114(3): 1021-29), chromoplastspecific carotenoid gene (oxidative stress; Capsicum; see Bouvier et al.(1998) J Biol Chem 273: 30651-59).

Promoters and control elements providing preferential transcriptionduring wounding or induced by methyl jasmonate can produce a defenseresponse in host cells or organisms. In a plant, for example,preferential modulation of genes, transcripts, and/or polypeptides undersuch conditions is useful to induce a defense response to mechanicalwounding, pest or pathogen attack or treatment with certain chemicals.

Promoters and control elements of the present invention also can triggera response similar to those described for cf9 (viral pathogen; tomato;see O'Donnell et al. (1998) Plant J 14(1): 137-42), hepatocyte growthfactor activator inhibitor type 1 (HAI-1), which enhances tissueregeneration (tissue injury; human; Koono et al. (1999) J HistochemCytochem 47: 673-82), copper amine oxidase (CuAO), induced duringontogenesis and wound healing (wounding; chick-pea; Rea et al. (1998)FEBS Lett 437: 177-82), proteinase inhibitor II (wounding; potato; seePena-Cortes et al. (1988) Planta 174: 84-89), protease inhibitor II(methyl jasmonate; tomato; see Farmer and Ryan (1990) Proc Natl Acad SciUSA 87: 7713-7716), two vegetative storage protein genes VspA and VspB(wounding, jasmonic acid, and water deficit; soybean; see Mason andMullet (1990) Plant Cell 2: 569-579).

Up-regulation and transcription down-regulation are useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease oxidative, flood, or drought tolerance may requireup-regulation of transcription.

Typically, promoter or control elements, which provide preferentialtranscription in wounding or under methyl jasmonate induction, producetranscript levels that are statistically significant as compared to celltypes, organs or tissues under other conditions.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

7.4. Light Induced Preferential Transcription

Promoters and control elements providing preferential transcription wheninduced by light exposure can be utilized to modulate growth,metabolism, and development; to increase drought tolerance; and decreasedamage from light stress for host cells or organisms. In a plant, forexample, modulation of genes, transcripts, and/or polypeptides inresponse to light is useful

-   -   (1) to increase the photosynthetic rate;    -   (2) to increase storage of certain molecules in leaves or green        parts only, e.g. silage with high protein or starch content;    -   (3) to modulate production of exogenous compositions in green        tissue, e.g. certain feed enzymes;    -   (4) to induce growth or development, such as fruit development        and maturity, during extended exposure to light;    -   (5) to modulate guard cells to control the size of stomata in        leaves to prevent water loss, or    -   (6) to induce accumulation of beta-carotene to help plants cope        with light induced stress.

The promoters and control elements of the present invention also cantrigger responses similar to those described in: abscisic acidinsensitive3 (ABI3) (dark-grown Arabidopsis seedlings, see Rohde et al.(2000) Plant Cell 12: 35-52), asparagine synthetase (pea root nodules,see Tsai and Coruzzi (1990) EMBO J 9: 323-32), mdm2 gene (human tumor,see Saucedo et al. (1998) Cell Growth Differ 9: 119-30).

Up-regulation and transcription down-regulation are useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease drought or light tolerance may require up-regulation oftranscription.

Typically, promoter or control elements, which provide preferentialtranscription in cells, tissues or organs exposed to light, producetranscript levels that are statistically significant as compared tocells, tissues, or organs under decreased light exposure (intensity orlength of time).

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

7.5. Dark Induced Preferential Transcription

Promoters and control elements providing preferential transcription wheninduced by dark or decreased light intensity or decreased light exposuretime can be utilized to time growth, metabolism, and development, tomodulate photosynthesis capabilities for host cells or organisms. In aplant, for example, modulation of genes, transcripts, and/orpolypeptides in response to dark is useful, for example,

-   -   (1) to induce growth or development, such as fruit development        and maturity, despite lack of light;    -   (2) to modulate genes, transcripts, and/or polypeptide active at        night or on cloudy days; or    -   (3) to preserve the plastid ultra structure present at the onset        of darkness.

The present promoters and control elements can also trigger responsesimilar to those described in the section above.

Up-regulation and transcription down-regulation is useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease or decrease growth and development may require up-regulation oftranscription.

Typically, promoter or control elements, which provide preferentialtranscription under exposure to dark or decrease light intensity ordecrease exposure time, produce transcript levels that are statisticallysignificant.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

7.6. Leaf Preferential Transcription

Promoters and control elements providing preferential transcription in aleaf can modulate growth, metabolism, and development or modulate energyand nutrient utilization in host cells or organisms. In a plant, forexample, preferential modulation of genes, transcripts, and/orpolypeptide in a leaf, is useful, for example,

(1) to modulate leaf size, shape, and development;

(2) to modulate the number of leaves; or

(3) to modulate energy or nutrient usage in relation to other organs andtissues

Up-regulation and transcription down-regulation is useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease growth, for example, may require up-regulation oftranscription.

Typically, promoter or control elements, which provide preferentialtranscription in the cells, tissues, or organs of a leaf, producetranscript levels that are statistically significant as compared toother cells, organs or tissues.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

7.7. Root Preferential Transcription

Promoters and control elements providing preferential transcription in aroot can modulate growth, metabolism, development, nutrient uptake,nitrogen fixation, or modulate energy and nutrient utilization in hostcells or organisms. In a plant, for example, preferential modulation ofgenes, transcripts, and/or polypeptide in a root, is useful,

-   -   (1) to modulate root size, shape, and development;    -   (2) to modulate the number of roots, or root hairs;    -   (3) to modulate mineral, fertilizer, or water uptake;    -   (4) to modulate transport of nutrients; or    -   (4) to modulate energy or nutrient usage in relation to other        cells, organs and tissues.

Up-regulation and transcription down-regulation is useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease or decrease growth, for example, may require up-regulation oftranscription.

Typically, promoter or control elements, which provide preferentialtranscription in cells, tissues, or organs of a root, produce transcriptlevels that are statistically significant as compared to other cells,organs or tissues.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

7.8. Stem/Shoot Preferential Transcription

Promoters and control elements providing preferential transcription in astem or shoot can modulate growth, metabolism, and development ormodulate energy and nutrient utilization in host cells or organisms. Ina plant, for example, preferential modulation of genes, transcripts,and/or polypeptide in a stem or shoot, is useful, for example,

(1) to modulate stem/shoot size, shape, and development; or

(2) to modulate energy or nutrient usage in relation to other organs andtissues

Up-regulation and transcription down-regulation is useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease growth, for example, may require up-regulation oftranscription.

Typically, promoter or control elements, which provide preferentialtranscription in the cells, tissues, or organs of a stem or shoot,produce transcript levels that are statistically significant as comparedto other cells, organs or tissues.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

7.9. Fruit and Seed Preferential Transcription

Promoters and control elements providing preferential transcription in asilique or fruit can time growth, development, or maturity; or modulatefertility; or modulate energy and nutrient utilization in host cells ororganisms. In a plant, for example, preferential modulation of genes,transcripts, and/or polypeptides in a fruit, is useful

-   -   (1) to modulate fruit size, shape, development, and maturity;    -   (2) to modulate the number of fruit or seeds;    -   (3) to modulate seed shattering;    -   (4) to modulate components of seeds, such as, storage molecules,        starch, protein, oil, vitamins, anti-nutritional components,        such as phytic acid;    -   (5) to modulate seed and/or seedling vigor or viability;    -   (6) to incorporate exogenous compositions into a seed, such as        lysine rich proteins;    -   (7) to permit similar fruit maturity timing for early and late        blooming flowers; or    -   (8) to modulate energy or nutrient usage in relation to other        organs and tissues.

Up-regulation and transcription down-regulation is useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease or decrease growth, for example, may require up-regulation oftranscription.

Typically, promoter or control elements, which provide preferentialtranscription in the cells, tissues, or organs of siliques or fruits,produce transcript levels that are statistically significant as comparedto other cells, organs or tissues.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

7.10. Callus Preferential Transcription

Promoters and control elements providing preferential transcription in acallus can be useful to modulating transcription in dedifferentiatedhost cells. In a plant transformation, for example, preferentialmodulation of genes, transcripts, in callus is useful to modulatetranscription of a marker gene, which can facilitate selection of cellsthat are transformed with exogenous polynucleotides.

Up-regulation and transcription down-regulation is useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease marker gene detectability, for example, may requireup-regulation of transcription.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

7.11. Flower Specific Transcription

Promoters and control elements providing preferential transcription inflowers can modulate pigmentation; or modulate fertility in host cellsor organisms. In a plant, for example, preferential modulation of genes,transcripts, and/or polypeptides in a flower, is useful,

(1) to modulate petal color; or

(2) to modulate the fertility of pistil and/or stamen.

Up-regulation and transcription down-regulation is useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease or decrease pigmentation, for example, may requireup-regulation of transcription

Typically, promoter or control elements, which provide preferentialtranscription in flowers, produce transcript levels that arestatistically significant as compared to other cells, organs or tissues.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

7.12. Immature Bud and Inflorescence Preferential Transcription

Promoters and control elements providing preferential transcription in aimmature bud or inflorescence can time growth, development, or maturity;or modulate fertility or viability in host cells or organisms. In aplant, for example, preferential modulation of genes, transcripts,and/or polypeptide in a immature bud and/or inflorescence, is useful,

(1) to modulate embryo development, size, and maturity;

(2) to modulate endosperm development, size, and composition;

(3) to modulate the number of seeds and fruits; or

(4) to modulate seed development and viability.

Up-regulation and transcription down-regulation is useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease or decrease growth, for example, may require up-regulation oftranscription.

Typically, promoter or control elements, which provide preferentialtranscription in immature buds and inflorescences, produce transcriptlevels that are statistically significant as compared to other celltypes, organs or tissues.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

7.13. Senescence Preferential Transcription

Promoters and control elements providing preferential transcriptionduring senescence can be used to modulate cell degeneration, nutrientmobilization, and scavenging of free radicals in host cells ororganisms. Other types of responses that can be modulated include, forexample, senescence associated genes (SAG) that encode enzymes thoughtto be involved in cell degeneration and nutrient mobilization(Arabidopsis; see Hensel et al. (1993) Plant Cell 5: 553-64), and theCP-2/cathepsin L gene (rat; Kim and Wright (1997) Biol Reprod 57:1467-77), both induced during senescence.

In a plant, for example, preferential modulation of genes, transcripts,and/or polypeptides during senescence is useful to modulate fruitripening.

Up-regulation and transcription down-regulation is useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease or decrease scavenging of free radicals, for example, mayrequire up-regulation of transcription.

Typically, promoter or control elements, which provide preferentialtranscription in cells, tissues, or organs during senescence, producetranscript levels that are statistically significant as compared toother conditions.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

7.14. Germination Preferential Transcription

Promoters and control elements providing preferential transcription in agerminating seed can time growth, development, or maturity; or modulateviability in host cells or organisms. In a plant, for example,preferential modulation of genes, transcripts, and/or polypeptide in agerminating seed, is useful,

(1) to modulate the emergence of the hypocotyls, cotyledons and radical;or

(2) to modulate shoot and primary root growth and development;

Up-regulation and transcription down-regulation is useful for theseapplications. For instance, genes, transcripts, and/or polypeptides thatincrease or decrease growth, for example, may require up-regulation oftranscription.

Typically, promoter or control elements, which provide preferentialtranscription in a germinating seed, produce transcript levels that arestatistically significant as compared to other cell types, organs ortissues.

For preferential up-regulation of transcription, promoter and controlelements produce transcript levels that are above background of theassay.

8. GFP EXPERIMENTAL PROCEDURES AND RESULTS Procedures

The polynucleotide sequences of the present invention were tested forpromoter activity using Green Fluorescent Protein (GFP) assays in thefollowing manner.

Approximately 1-3 kb of genomic sequence occurring immediately upstreamof the ATG translational start site of the gene of interest was isolatedusing appropriate primers tailed with BstXI restriction sites. StandardPCR reactions using these primers and genomic DNA were conducted. Theresulting product was isolated, cleaved with BstXI and cloned into theBstXI site of an appropriate vector, such as pNewBin4-HAP1-GFP (see FIG.1).

Agrobacterium-Mediated Transformation of Arabidopsis

Host Plants and Transgenes: Wild-type Arabidopsis thaliana Wassilewskija(WS) plants are transformed with Ti plasmids containing nucleic acidsequences to be expressed, as noted in the respective examples, in thesense orientation relative to the 35S promoter in a Ti plasmid. A Tiplasmid vector useful for these constructs, CRS 338, contains theCeres-constructed, plant selectable marker gene phosphinothricinacetyltransferase (PAT), which confers herbicide resistance totransformed plants.

Ten independently transformed events are typically selected andevaluated for their qualitative phenotype in the T₁ generation.

Preparation of Soil Mixture: 24 L Sunshine Mix #5 soil (Sun GroHorticulture, Ltd., Bellevue, Wash.) is mixed with 16 L Therm-O-Rockvermiculite (Therm-O-Rock West, Inc., Chandler, Ariz.) in a cement mixerto make a 60:40 soil mixture. To the soil mixture is added 2 TbspMarathon 1% granules (Hummert, Earth City, Mo.), 3 Tbsp OSMOCOTE®14-14-14 (Hummert, Earth City, Mo.) and 1 Tbsp Peters fertilizer20-20-20 (J.R. Peters, Inc., Allentown, Pa.), which are first added to 3gallons of water and then added to the soil and mixed thoroughly.Generally, 4-inch diameter pots are filled with soil mixture. Pots arethen covered with 8-inch squares of nylon netting.

Planting: Using a 60 mL syringe, 35 mL of the seed mixture is aspirated.25 drops are added to each pot. Clear propagation domes are placed ontop of the pots that are then placed under 55% shade cloth andsubirrigated by adding 1 inch of water.

Plant Maintenance: 3 to 4 days after planting, lids and shade cloth areremoved. Plants are watered as needed. After 7-10 days, pots are thinnedto 20 plants per pot using forceps. After 2 weeks, all plants aresubirrigated with Peters fertilizer at a rate of 1 Tsp per gallon ofwater. When bolts are about 5-10 cm long, they are clipped between thefirst node and the base of stem to induce secondary bolts. Dippinginfiltration is performed 6 to 7 days after clipping.

Preparation of Agrobacterium: To 150 mL fresh YEB is added 0.1 mL eachof carbenicillin, spectinomycin and rifampicin (each at 100 mg/ml stockconcentration). Agrobacterium starter blocks are obtained (96-well blockwith Agrobacterium cultures grown to an OD₆₀₀ of approximately 1.0) andinoculated one culture vessel per construct by transferring 1 mL fromappropriate well in the starter block. Cultures are then incubated withshaking at 27° C. Cultures are spun down after attaining an OD₆₀₀ ofapproximately 1.0 (about 24 hours). 200 mL infiltration media is addedto resuspend Agrobacterium pellets. Infiltration media is prepared byadding 2.2 g MS salts, 50 g sucrose, and 5 μL 2 mg/ml benzylaminopurineto 900 ml water.

Dipping Infiltration: The pots are inverted and submerged for 5 minutesso that the aerial portion of the plant is in the Agrobacteriumsuspension. Plants are allowed to grow normally and seed is collected.

High-throughput Screening of T₁ Transgenic Plants: Seed is evenlydispersed into water-saturated soil in pots and placed into a dark 4° C.cooler for two nights to promote uniform germination. Pots are thenremoved from the cooler and covered with 55% shade cloth for 4-5 days.Cotyledons are fully expanded at this stage. FINALE® (Sanofi Aventis,Paris, France) is sprayed on plants (3 ml FINALE® diluted into 48 oz.water) and repeated every 3-4 days until only transformants remain.

GFP Assay

Tissues are dissected by eye or under magnification using INOX 5 gradeforceps and placed on a slide with water and coversliped. An attempt ismade to record images of observed expression patterns at earliest andlatest stages of development of tissues listed below. Specific tissueswill be preceded with High (H), Medium (M), Low (L) designations.

Flower Pedicel, receptacle, nectary, sepal, petal, filament, anther,pollen, carpel, style, papillae, vascular, epidermis, stomata, trichomeSilique Stigma, style, carpel, septum, placentae, transmitting tissue,vascular, epidermis, stomata, abscission zone, ovule OvulePre-fertilization: inner integument, outer integument, embryo sac,funiculus, chalaza, micropyle, gametophyte Post-fertilization: zygote,inner integument, outer integument, seed coat, primordia, chalaza,micropyle, early endosperm, mature endosperm, embryo Embryo Suspensor,preglobular, globular, heart, torpedo, late mature, provascular,hypophysis, radicle, cotyledons, hypocotyl Stem Epidermis, cortex,vascular, xylem, phloem, pith, stomata, trichome Leaf Petiole,mesophyll, vascular, epidermis, trichome, primordia, stomata, stipule,margin

T1 Mature: These are the T1 plants resulting from independenttransformation events. These are screened between stage 6.50-6.90 (i.e.the plant is flowering and 50-90% of the flowers that the plant willmake have developed), which is 4-6 weeks of age. At this stage themature plant possesses flowers, siliques at all stages of development,and fully expanded leaves. The plants are initially imaged under UV witha Leica Confocal microscope to allow examination of the plants on aglobal level. If expression is present, they are re-imaged usingscanning laser confocal microscopy.

T2 Seedling: Progeny are collected from the T1 plants giving the sameexpression pattern and the progeny (T2) are sterilized and plated onagar-solidified medium containing M&S salts. In the event that there isno expression in the T1 plants, T2 seeds are planted from all lines. Theseedlings are grown in Percival incubators under continuous light at 22°C. for 10-12 days. Cotyledons, roots, hypocotyls, petioles, leaves, andthe shoot meristem region of individual seedlings were screened untiltwo seedlings were observed to have the same pattern. In general, thesame expression pattern was found in the first two seedlings. However,up to 6 seedlings were screened before “no expression pattern” wasrecorded. All constructs are screened as T2 seedlings even if they didnot have an expression pattern in the T1 generation.

T2 Mature: The T2 mature plants were screened in a similar manner to theT1 plants. The T2 seeds were planted in the greenhouse, exposed toselection and at least one plant screened to confirm the T1 expressionpattern. In instances where there were any subtle changes in expression,multiple plants were examined and the changes noted in the tables.

T3 Seedling: This was done similar to the T2 seedlings except that onlythe plants for which we are trying to confirm the pattern are planted.

Image Data:

Images are collected by scanning laser confocal microscopy. Scannedimages are taken as 2-D optical sections or 3-D images generated bystacking the 2-D optical sections collected in series. All scannedimages are saved as TIFF files by imaging software, edited in AdobePhotoshop, and labeled in Powerpoint specifying organ and specificexpressing tissues.

Results

The Promoter Expression Reports of the Tables present the results of theGFP assays as reported by their corresponding construct number and linenumber.

Promoter Expression Report For PT0960 (SEQ ID NO: 1) Promoter Tested In:Arabidopsis thaliana, Wassilewskija (WS) ecotype Spatial expressionsummary: Hypocotyl H vascular Cotyledon H vascular Rosette Leaf Hvascular Primary Root L epidermis L cortex H endodermis H vascularObserved expression pattern: T1 Mature expression: None observed. T2Seedling expression: High GFP expression throughout vasculature ofseedlings. Source Promoter Organism: Arabidopsis thaliana, Columbia(Col) ecotype Vector: pNewbin4-HAP1-GFP Marker Type: GFP-ER GenerationScreened: XT1 Mature XT2 Seedling Inductions completed. Events Screened/Treatment: Age: Gen: Time points: Response Response: 1. 14.3 mM KNO₃ to28.6 4 wks T2 72 hrs post 2/0 No Mannitol transfer

TABLE 1-1 T1 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 3 Events Expressing: n = 0 No GFP Expression Detected

TABLE 2-1 T2 Seedline Expression Tissues Screened Events Screened: n = 3Events Expressing: n = 3 Expression Detected Hypocotyl H vascularCotyledon H vascular Rosette Leaf H vascular Primary Root L epidermis Lcortex H endodermis H vascular

Construct: PT0960 Promoter candidate I.D: 22254785 cDNA I.D: 23518786Events expressing: 01-03

Promoter Expression Report YP2585 (SEQ ID NO: 2) deletion of PT0743Promoter Tested In: Arabidopsis thaliana, Wassilewskija (WS) ecotypeSpatial expression summary: Primary Root H epidermis H cortex H roothairs Mature Root H epidermis H vascular H pericycle H stele Observedexpression pattern: T1 Mature expression: No expression detected T2Seedling expression: Expression in root epidermis and cortex T2 Matureexpression: No expression detected Source Promoter Organism: Arabidopsisthaliana, Columbia (Col) ecotype Vector: pNewbin4-HAP1-GFP Marker Type:GFP-ER Generation Screened: X T1 Mature X T2 Seedling X T2 Mature

TABLE 1-2 T1 Mature Plant Expression Organs/Tissues screened No GFPExpression Detected

TABLE 2-2 T2 Seedling Expression Tissues Screened Events Screened: n = 6Events Expressing: n = 5 (02-06) Expression Detected Primary Root Hepidermis H cortex H root hairs

TABLE 3-2 T2 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 6 Events Expressing: n = 3 (03, 04, 06 Expression DetectedMature Root H epidermis H vascular H pericycle H stele

TABLE 4-2 Promoter utility Trait Area: Water Use Efficiency, NutrientUse Efficiency Sub-trait Area: Drought Tolerance Utility: Among otheruses this promoter sequence is useful to improve the uptake of water andnutrients from the soil.

Construct: YP2585 Promoter candidate I.D: 40983033 cDNA I.D: 23509083Events expressing: 02-06One or more fragments of the above described promoter are identified inthe miscellaneous feature section of the relevant SEQ ID in the SequenceListing. Those fragments were tested for promoter activity by the sameprocedures as described above, and the results are summarized below. Noexpression is observed for Ceres Promoter YP2581.

Promoter Expression Report For PT0998 (SEQ ID NO: 3) Promoter Tested In:Arabidopsis thaliana, Wassilewskija (WS) ecotype Spatial expressionsummary: Flower H anther H tapetum H silique Silique L vascular Lepidermis Mature root H mature root Primary Root H cortex H endodermis Hvascular L xylem H phloem H pericycle Observed expression pattern: T1Mature expression: High GFP expression in roots, tapetum cells ofdeveloping anthers and siliques. T2 Seedling expression: High GFPexpression in cortex, endodermis, and surrounding vascular bundle. T2Mature expression: High GFP expression in roots (vasculature) of matureplants. Source Promoter Organism: Arabidopsis thaliana, Columbia (Col)ecotype Vector: pNewbin4-HAP1-GFP Marker Type: GFP-ER GenerationScreened: X T1 Mature X T2 Seedling X T2 Mature

TABLE 1-3 T1 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 4 Events Expressing: n = 4 Expression Detected Flower Hanther H tapetum H silique H Silique L vascular L epidermis Root H Yes

TABLE 2-3 T2 Seedling Expression Tissues Screened Events Screened: n = 4Events Expressing: n = 4 GFP Expression Detected X Primary Root H cortexH endodermis H vascular L xylem H phloem H pericycle

TABLE 3-3 T2 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 4 Events Expressing: n = 4 Expression Detected Mature RootH mature root

TABLE 4-3 RT-PCR Results: Plants were grown hydroponically tor 4 weeksuntil tissue collection. Roots and aerial tissues (Aerials) wereharvested separately in liquid nitrogen. For each event, two sets ofsamples are collected for qRT-PCR analysis. The data presented is theaverage ratio of roots and aerials between two replicates. PT0998-2PT0998-3 PT0998-4 Ratio Ratio Ratio (Roots/Aerials) (Roots/Aerials)(Roots/Aerials) HAP 6.33 23.16 33.01 GFP 253.98 575.31 555.41

TABLE 5-3 Promoter utility Trait Area: Stress, Nutrients Sub-trait Area:nitrogen utilization, drought tolerance, UV-B tolerance Utility: Amongother uses this promoter sequence is useful to improve: Modulatenutrients, water uptake from soil and transportation within plants.Provide drought or UV protection.

Construct: PT0998 Promoter candidate I.D: 24469840 cDNA I.D: 23506262 +23545255 Events expressing: 01, 02, 03, 04One or more fragments of the above described promoter are identified inthe miscellaneous feature section of the relevant SEQ ID in the SequenceListing. Those fragments were tested for promoter activity by the sameprocedures as described above, and the results are summarized below. Noexpression is observed for Ceres Promoter PD3457.

Promoter Expression Report YP2219 (SEQ ID NO: 4) Promoter Tested In:Arabidopsis thaliana, Wassilewskija (WS) ecotype Spatial expressionsummary: Hypocotyl L epidermis L cortex L vascular Cotyledon L hydathodeL petiole L epidermis Rosette Leaf L epidermis H petiole Primary Root Lepidermis H cortex H vascular Lateral root H lateral root cap Observedexpression pattern: T1 Mature expression: No GFP expression observed T2Seedling expression: Hypocotyl, cotyledon, rosette leaf, primary, andlateral root T2 Mature expression: Source Promoter Organism: Arabidopsisthaliana, Columbia (Col) ecotype Vector: pNewbin4-HAP1-GFP Marker Type:GFP-ER Generation Screened: X T1 Mature X T2 Seedling Inductionscompleted. Events Screened/ Treatment: Age: Gen: Time points: ResponseResponse: 1. Drought 4 wks T2 1.0% moisture 6/4 Yes Inducible expressionsummary: Treatment: Time point induced: Organs induced: Tissuesinduced: 1. Drought 1.0% moisture Stem, leaf, leaf

TABLE 1-4 T1 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 6 Events Expressing: n = 0 No GFP Expression Detected

TABLE 2-4 T2 Seedling Expression Tissues Screened Events Screened: n = 6Events Expressing: n = 4 Expression Detected Hypocotyl L epidermis Lcortex L vascular Cotyledon L epidermisd L petiole L hydathode RosetteLeaf L epidermis H petiole Primary Root L epidermis H cortex H vascularLateral root H lateral root cap

TABLE 4-4 Promoter utility Trait Area: Water Use Efficiency Sub-traitArea: Drought Tolerance Utility: Among other uses this promoter sequenceis useful to engineer drought tolerance in plants.

Construct: YP2219 Promoter candidate I.D: 37172464 cDNA I.D: 23494283Events expressing: 01, 02, 04, 06One or more fragments of the above described promoter are identified inthe miscellaneous feature section of the relevant SEQ ID in the SequenceListing. Those fragments were tested for promoter activity by the sameprocedures as described above, and the results are summarized below. Noinduction is observed for Ceres Promoter YP2229.

Promoter Expression Report For YP0286 (SEQ ID NO: 5) Promoter Tested In:Arabidopsis thaliana, Wassilewskija (WS) ecotype Spatial expressionsummary: Flower L pedicel L epidermis Stem L epidermis Hypocotyl Hepidermis Cotyledon H mesophyll H vascular H epidermis H petiole RosetteLeaf H epidermis H petiole Primary Root H epidermis Lateral root Hlateral root cap Observed expression pattern: T1 mature: Low epidermalexpression in stem and pedicles near inflorescence apical meristem. T2seedling: High epidermal expression in cotyledons, petioles of emergingrosette leaves, hypocotyl, and root. Expression observed in vascular andmesophyll cells of cotyledons. Source Promoter Organism: Arabidopsisthaliana, WS ecotype Vector: pNewbin4-HAP1-GFP Marker Type: GFP-ERGeneration Screened: XT1 Mature XT2 Seedling Inductions completed:Events Screened/ Treatment: Age: Gen: Time points: Response:Response: 1. Drought 7 days T2 3 Hrs Air dry 2/0 No 2. Drought 4 weeksT2 10-12 day No H20 2/2 Yes Inducible expression summary: Treatment:Time point induced: Organs induced: Tissues induced: 2. Drought 10-12day No H20 Flowers Pedicel, Epidermis Siliques Epidermis Leaf Epidermis,Vascular Stem Epidermis

TABLE 1-5 T1 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 6 Events Expressing: n = 2 GFP Expression Detected FlowerL pedicel L epidermis Stem L epidermis

TABLE 2-5 T2 Seedling Expression Tissues Screened Events Screened: n = 2Events Expressing: n = 2 Seedlings expressing/Seedlings screenedEvent-04: 6/6 Event-06: 4/6 Expression Detected Hypocotyl H epidermisCotyledon H mesophyll H vascular H epidermis H petiole Rosette Leaf Hepidermis H petiole Primary Root H epidermis Lateral root H lateral rootcap

TABLE 3-5 Promoter utility Trait Area: Water use efficiency Sub-traitArea: Drought Utility: Among other uses this promoter sequence is usefulto improve: Modulation growth and development. Modulation of nutrientuptake and loading. Expression of nitrate transports and water pumps.Modulation of drought responses, including modulation of water uptakeand transport under drought conditions. Notes: Candidate to drive genesinvolved in osmotic stresses such as NCED. Endogenous promoter inducedunder drought.

Construct: YP0286 Promoter candidate I.D: 11768589 cDNA I.D: 12669548(OCKHAM3-C) Lines expressing: YP0286 -04, -06; 7/3/03

Promoter Expression Report YP1692 (SEQ ID NO: 6) Promoter Tested In:Arabidopsis thaliana, Wassilewskija (WS) ecotype Spatial expressionsummary: Flower L petal L vascular Hypocotyl L epidermis L vascularCotyledon L vascular Primary Root H vascular Lateral root H vascularRoot H mature root Observed expression pattern: T1 mature expression:GFP expressed in vasculature of petals in flowers. T2 seedlingexpression: High GFP expression in vasculature of root. Low GFPexpression in hypocotyl and cotyledons. GFP expressed in epidermis ofhypocotyl near root transition zone, T1 mature expression: GFP expressedin vasculature of root. Source Promoter Organism: Arabidopsis thaliana,Columbia (Col) ecotype Vector: pNewbin4-HAP1-GFP Marker Type: GFP-ERGeneration Screened: X T1 Mature X T2 Seedling X T2 Mature Inductionscompleted. Events Screened/ Treatment: Age: Gen: Time points: ResponseResponse: 1. Drought 4 wks T2 1.0% moisture 6/2 Yes Inducible expressionsummary: Treatment: Time point induced: Organs induced: Tissues induced:

TABLE 1-6 T1 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 6 Events Expressing: n = 3 Expression Detected Flower Lpetal L vascular

TABLE 2-6 T2 Seedling Expression Tissues Screened Events Screened: n = 6Events Expressing: n = l Expression Detected Hypocotyl L epidermis Lvascular Cotyledon L vascular Primary Root H vascular Lateral root Hvascular

TABLE 3-6 T2 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 5 Events Expressing: n = 2 Expression Detected X Root Hmature root

Construct: YP1692 Promoter candidate I.D: 15371608 cDNA I.D: 36534367Events expressing: 01-06

Promoter Expression Report YP1894 (SEQ ID NO: 7) Promoter Tested In:Arabidopsis thaliana, Wassilewskija (WS) ecotype Spatial expressionsummary: Cotyledon L epidermis Primary Root H epidermis H cortex Root Hmature root Observed expression pattern: Root Specific. T1 Matureexpression: No expression detected. T2 Seedling expression: High GFPexpression in epidermis and cortex cells of root. Low GFP expression incotyledons. T2 Mature expression: High GFP expression in roots.Inductions: Expression enhanced by high-nitrogen conditions (seeInduction table below). Source Promoter Organism: Arabidopsis thaliana,Columbia (Col) ecotype Vector: pNewbin4-HAP1-GFP Marker Type: GFP-ERGeneration Screened: X T1 Mature X T2 Seedling X T2 Mature InductionsTable. Upregulation of gene expression was expected in low-nitrogenconditions. This was not observed. However, enhanced GFP was detected inresponse to the high-nitrogen condition (control test) (see Table 4below). Events Time Screened/ Treatment: Age: Gen: points: ResponseResponse: 1. Nitrogen (High to Low)— 4 wks T2 90 hrs 6/0 No 14.3 mM KNO₃to 28.6 mM Mannitol Inducible expression summary: Treatment: Time pointinduced: Organs induced: Tissues induced: High nitrogen 90 hrs Leavesand stems

TABLE 1-7 T1 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 6 Events Expressing: n = 0 No GFP Expression Detected

TABLE 2-7 T2 Seedling Expression Tissues Screened Events Screened: n = 6Events Expressing: n = 6(01-06) Expression Detected Cotyledon Lepidermis Primary Root H epidermis H cortex

TABLE 3-7 T2 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 6 Events Expressing: n = 6 (01-06) Expression DetectedRoot H mature root

TABLE 5-7 Promoter utility Trait Area: Water Use Efficiency, NutrientSub-trait Area: Drought, Nitrogen use efficiency, Utility: Among otheruses this promoter sequence is useful to improve: Expression in theroots is useful for improving water and nutrient uptake into the rootmass without undesirable affects on the above ground tissues. Thehigh-nitrogen induction is useful in reducing the toxicity associatedwith very high levels of nitrogen and/or bypassing the feedback loopthat limits nitrogen uptake when nitrogen levels are not limiting, thusaccelerating nitrogen uptake for either storage for later use or forenhancing growth.

Construct: YP1894 Promoter candidate I.D: 25518825 cDNA I.D: 23499704

Promoter Expression Report For YP1976 (SEQ ID NO: 8) Promoter Tested In:Arabidopsis thaliana, Wassilewskija (WS) ecotype Spatial expressionsummary: Hypocotyl L epidermis Primary Root H cortex Lateral root Hcortex Root H mature root Observed expression pattern: T1 matureexpression: No GFP expression observed in aerial organs. T2 seedlingexpression: High GFP expressed in epidermal cells at root transitionzone. GFP is expressed in cortex cells of lower main root and lateralroot. T2 mature expression: High GFP expression in roots of matureplant. No GFP expression observed in aerial tissues. Source PromoterOrganism: Arabidopsis thaliana, Columbia (Col) ecotype Vector:pNewbin4-HAP1-GFP Marker Type: GFP-ER Generation Screened: X T1 Mature XT2 Seedling X T2 Mature

TABLE 1-8 T1 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 6 Events Expressing: n = 0 No GFP Expression Detected

TABLE 2-8 T2 Seedling Expression Tissues Screened Events Screened: n = 6Events Expressing: n = 6 Expression Detected X Hypocotyl L epidermis XPrimary Root H cortex X Lateral root H cortex

TABLE 3-8 T2 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 6 Events Expressing: n = 6 Expression Detected X Root Hmature root

TABLE 4-8 Promoter utility Trait Area: Nutrient and water economy.Sub-trait Area: Nitrogen use efficiency Water use efficiency. Utility:Among other uses this promoter sequence is useful to improve: Nitrogenuse efficiency in lower/non-limiting nitrogen environments, enhancedwater uptake in drought and non-limiting water environments, andprotection against soil-borne nematodes, root worms, fungal andbacterial pathogens.

Construct: YP1976 Promoter candidate I.D: 15371806 cDNA I.D: 23523729Events expressing: 01-06

Promoter Expression Report YP2016 (SEQ ID NO: 9) Promoter Tested In:Arabidopsis thaliana, Wassilewskija (WS) ecotype Spatial expressionsummary: Flower H pedicel H receptacle H nectary H sepal H petal Hfilament H tapetum H carpel H style H papillae H vascular H epidermis Hstomata H silique Silique H stigma H style H carpel H septum H vascularH epidermis H abscission zone H ovule Ovule Post-fertilization: H innerintegument H outer integument H seed coat H embryo Embryo H torpedo Hlate H mature Stem H vascular H xylem H phloem H pith H epidermis Hcortex Leaf H petiole H mesophyll H epidermis H stomata Hypocotyl Hepidermis H cortex H vascular H xylem H phloem Cotyledon H mesophyll Hvascular H epidermis H petiole Rosette Leaf H mesophyll H vascular Hepidermis Primary Root H cortex H vascular Lateral root H root capObserved expression pattern: T1 Mature expression: GFP broadly expressedthroughout mature plant with highest expression at inflorescencemeristems. High GFP expression throughout organs of flowers. Notexpressed in anther walls of mature stamen. High GFP expression insilique, developing ovules, seed and embryos. GFP expression inepidermis, vasculature and mesophyll in leaves. GFP expressed inepidermis, cortex, pith, and vascular bundles throughout stem, highestnear apex decreasing toward rosette leaves. GFP expression in matureroot. T2 Seedling expression: GFP expression throughout epidermis,cortex, vascular and mesophyll cells in aerial tissues of seedling. Inroot, GFP expressed in cortex and vasculature. T2 Mature expression: GFPbroadly expressed throughout mature plant with highest expression atinflorescence meristems and root. Source Promoter Organism: Arabidopsisthaliana, Columbia (Col) ecotype Vector: pNewbin4-HAP1-GFP Marker Type:GFP-ER Generation Screened: X T1 Mature X T2 Seedling X T2 Mature

TABLE 1-9 T1 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 6 Events Expressing: n = 6 Expression Detected Flower Hpedicel H receptacle H nectary H sepal H petal H filament H tapetum Hcarpel H style H papillae H vascular H epidermis H stomata H siliqueSilique H stigma H style H carpel H septum H vascular H epidermis Habscission zone H ovule Ovule Post-fertilization: H inner integument Houter integument H seed coat H embryo Embryo H torpedo H late H matureStem H epidermis H cortex H vascular H xylem H phloem H pith Leaf Hpetiole H mesophyll H epidermis H stomata

TABLE 2-9 T2 Seedling Expression Tissues Screened Events Screened: n = 4Events Expressing: n = 4 Expression Detected Hypocotyl H epidermis Hcortex H vascular H xylem H phloem Cotyledon H mesophyll H vascular Hepidermis H petiole Rosette Leaf H mesophyll H vascular H epidermisPrimary Root H cortex H vascular Lateral root H root cap

TABLE 3-9 T2 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 4 Events Expressing: n = 4

TABLE 4-9 Promoter utility Trait Area: Plant growth and developmentSub-trait Area: Size and source capacity Utility: Among other uses thispromoter sequence is useful to improve: Plant size and architecture,growth rate, seedling establishment, responses to shade and low light,responses to drought and cold, source capacity and sucrose loading, seedfilling, seed size and plant yield.

Construct: YP2016 Promoter candidate I.D: 25087074 cDNA I.D: 23495629Events expressing: 01-04

Promoter Expression Report YP2097 (SEQ ID NO: 10) Promoter Tested In:Arabidopsis thaliana, Wassilewskija (WS) ecotype Spatial expressionsummary: Flower H pollen Primary Root L epidermis Root L mature rootObserved expression pattern: T1 mature expression: High GFP expressionspecific to pollen. T2 seedling expression: GFP expression in epidermalcells at root transition zone decreasing toward root tip. T2 matureexpression: High GFP expression in pollen. GFP expression in epidermisand vascular cells of root. Source Promoter Organism: Arabidopsisthaliana, Columbia (Col) ecotype Vector: pNewbin4-HAP1-GFP Marker Type:GFP-ER Generation Screened: X T1 Mature X T2 Seedling X T2 MatureInductions completed. Events Screened/ Treatment: Age: Gen: Time points:Response Response: 1. Drought 4 wks T2 1.0% moisture 6/2 Yes Inducibleexpression summary: Treatment: Time point induced: Organs induced:Tissues induced: 1. Drought 1.0% moisture Flower Pollen, Epidermis LeafGuard cells, Epidermis Stem Guard cells Root Vascular

TABLE 1-10 T1 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 6 Events Expressing: n = 4 Expression Detected Flower Hpollen

TABLE 2-10 T2 Seedling Expression Tissues Screened Events Screened: n =6 Events Expressing: n = 2 Expression Detected Primary Root L epidermis

TABLE 3-10 T2 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 6 Events Expressing: n = 3 Expression Detected Root Lmature root

TABLE 5-10 Promoter utility Trait Area: PG&D, water use efficiencySub-trait Area: Drought tolerance, Utility: Among other uses thispromoter sequence is useful to improve: Desiccation tolerance, recoveryfrom drought, drought tolerance, improve water use efficiency, seed sizeand nutrient use efficiency and nitrogen use efficiency.

Construct: YP2097 Promoter candidate I.D: 29223804 cDNA I.D: 36541759Events expressing: 02, 04

Promoter Expression Report YP2538 (SEQ ID NO: 11) Promoter Tested In:Arabidopsis thaliana, Wassilewskija (WS) ecotype Spatial expressionsummary: Flower H pedicel H anther H silique Silique H carpel Hepidermis Ovule Post-fertilization: H embryo Embryo H mature Hypocotyl Hepidermis H vascular Cotyledon H mesophyll H vascular H epidermisRosette Leaf H mesophyll H epidermis Primary Root H vascular H root capAerial organs H inflorescence H flowers H silique H stem Root H matureroot Observed expression pattern: T1 Mature expression: High GFPexpression at inflorescence. GFP expressed in stem and pedicels nearapex. High GFP expression in anthers of developing flowers and incarpels and mature embryos in mature siliques. T2 Seedling expression:High GFP expression in vasculature of root, hypocotyl and cotyledons.High GFP expression in epidermis, mesophyll and vasculature ofcotyledons. High GFP expression in emerging rosette leaves. T2 Matureexpression: High GFP expression at inflorescence, flowers, stem androot. Source Promoter Organism: Arabidopsis thaliana, Columbia (Col)ecotype Vector: pNewbin4-HAP1-GFP Marker Type: GFP-ER GenerationScreened: X T1 Mature X T2 Seedling X T2 Mature Inductions completed.Events Screened/ Treatment: Age: Gen: Time points: Response Response: 1.Drought 4 wks T2 1.0% moisture 4/3 Yes Inducible expression summary:Treatment: Time point induced: Organs induced: Tissues induced: 1.Drought 1.0% moisture Inflorescence Pedicels, Silique, Stem FlowerSilique, Stamens Silique Epidermis, Vascular Stem Vascular RootEpidermis

TABLE 1-11 T1 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 5 Events Expressing: n = 2 (02, 05) Expression DetectedFlower H pedicel H anther H silique Silique H carpel H epidermis OvulePost-fertilization: H embryo Embryo H mature

TABLE 2-11 T2 Seedling Expression Tissues Screened Events Screened: n =3 Events Expressing: n = 2 (03, 05) Expression Detected Hypocotyl Hepidermis H vascular Cotyledon H mesophyll H vascular H epidermisRosette Leaf H mesophyll H epidermis Primary Root H vascular H root cap

TABLE 3-11 T2 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 4 Events Expressing: n = 2 (03, 05) Expression DetectedAerial organs H inflorescence H flowers H silique H stem Root H matureroot

TABLE 5-11 Promoter utility Trait Area: Water Use Efficiency, NutrientUse Efficiency, Yield Sub-trait Area: Drought Tolerance, Low NitrogenTolerance, Low Phosphorous Tolerance, Seed Size and Yield Utility: Amongother uses this promoter sequence is useful to improve: response todrought conditions and low soil nutrient levels. Expression in theembryo could be valuable for engineering of seed size and yield.

Construct: YP2538 Promoter candidate I.D: 25087125 cDNA I.D: 23519856Events expressing: 03-05

Promoter Expression Report YP2552 (SEQ ID NO: 12) Promoter Tested In:Arabidopsis thaliana, Wassilewskija (WS) ecotype Spatial expressionsummary: Primary Root H epidermis H vascular H root hairs Mature Root Hepidermis H vasculature H xylem H phloem H root hairs lateral root Lstele Observed expression pattern: T1 Mature expression: No GFPexpression detected. T2 Seedling expression: Root specific GFPexpression. High GFP expression in epidermal and vascular cells. T2Mature expression: Root specific GFP expression. High GFP expression inepidermal and vascular cells. Source Promoter Organism: Arabidopsisthaliana, Columbia (Col) ecotype Vector: pNewbin4-HAP1-GFP Marker Type:GFP-ER Generation Screened: X T1 Mature X T2 Seedling X T2 MatureInductions completed: Events Screened/ Treatment: Age: Gen: Time points:Response Response: 1. Cold 10 day T2 4 hr 6/0 None 2. Nitrogen—high N tolow N  4 weeks T2 4 hr 6/0 None [14.3 mM KNO₃ to 28.6 mM Mannitol] 3.Far Red Far Red₇₃₀ = 525 10 day T2 4 hr 6/0 None μW/cm Inducibleexpression summary: Treatment: Time point induced: Organs induced:Tissues induced:

TABLE 1-12 T1 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 6 Events Expressing: n = 0 No GFP Expression Detected

TABLE 2-12 T2 Seedling Expression Tissues Screened Events Screened: n =6 Events Expressing: n = 4 (01-03, 06) Expression Detected X PrimaryRoot H epidermis H vasculature H root hairs

TABLE 3-12 T2 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 6 Events Expressing: n = 5 (01, 02, 03, 05, 06) ExpressionDetected Mature Root H epidermis H vasculature H xylem H phloem H roothairs lateral root L stele

TABLE 4-12 Promoter utility Trait Area: Water Use Efficiency, NutrientUse Efficiency Sub-trait Area: Drought Tolerance, Nitrogen andPhosphorous Use Efficiency Utility: Among other uses this promotersequence is useful to improve the uptake of water and nutrients.

Construct: YP2552 Promoter candidate I.D: 25659462 cDNA I.D: 23504306Events expressing: 01, 02, 03, 05, 06

Promoter Expression Report YP2563 (SEQ ID NO: 13) Promoter Tested In:Arabidopsis thaliana, Wassilewskija (WS) ecotype Spatial expressionsummary: Flower H carpel H silique Silique H carpel OvulePost-fertilization: H funiculus H seed coat Hypocotyl H epidermis Lvascular Cotyledon L epidermis H mesophyll H epidermis Primary Root Lepidermis L cortex H vascular H root cap H root hairs Lateral root Lepidermis Observed expression pattern: T1 Mature expression: High GFPexpression in carpels and in seed coats and funiculus of maturesiliques. T2 Seedling expression: High GFP expression in vasculaturethroughout root and in epidermis and cortex cells near root tip atelongation zone. GFP expressed in root hair and root cap. T2 Matureexpression: High GFP expression in roots and lateral inflorescences.Source Promoter Organism: Arabidopsis thaliana, Columbia (Col) ecotypeVector: pNewbin4-HAP1-GFP Marker Type: GFP-ER Generation Screened: X T1Mature X T2 Seedling X T2 Mature Inductions completed. Events Screened/Treatment: Age: Gen: Time points: Response Response: 1. Nitrogen—high Nto low N 4 wks T2 72 Hr 12/0 None [14.3 mM KNO₃ to 28.6 mM Mannitol]Inducible expression summary: Treatment: Time point induced: Organsinduced: Tissues induced:

TABLE 1-13 T1 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 12 Events Expressing: n = 12 (01-12) Expression DetectedFlower H carpel H silique Silique H carpel Ovule Post-fertilization: Hfuniculus H seed coat

TABLE 2-13 T2 Seedling Expression Tissues Screened Events Screened: n =11 Events Expressing: n = 9 (02-04, 06, 09-11, 13, 14) ExpressionDetected Hypocotyl H epidermis L vascular Cotyledon L epidermis RosetteLeaf H mesophyll H epidermis Primary Root L epidermis L cortex Hvascular H root cap H root hairs Lateral root L epidermis

TABLE 3-13 T2 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 5 Events Expressing: n = 4 (11-15) Expression DetectedAerial organs H inflorescence L stem Root H mature root

TABLE 4-13 Promoter utility Trait Area: Nutrient Use Efficiency, WaterUse Efficiency, Yield, PG&D, Confinement Sub-trait Area: Nitrogen andPhosphorous Utilization, Drought Tolerance, Seed Size, PlantEstablishment, Seed Confinement Utility: Among other uses this promotersequence could be useful to improve enhance the uptake of nutrients andwater from the soil (root), and seed size (seed coat). Expression in theseed could also be used to engineer seed ablation and seed confinement.

Construct: YP2563 Promoter candidate I.D: 25518834 cDNA I.D: 36516796Events expressing: 01-12

Promoter Expression Report YP2571 (SEQ ID NO: 14) Promoter Tested In:Arabidopsis thaliana, Wassilewskija (WS) ecotype Spatial expressionsummary: Flower H sepal H petal H anther H vascular H stomata H siliqueSilique H style H carpel H funiculus H vascular Leaf L vascular Aerialorgans H inflorescence Root H mature root Hypocotyl H vascular CotyledonH mesophyll H vascular H epidermis Primary Root H vascular Observedexpression pattern: T1 Mature expression: High GFP expression detectedin inflorescences. T2 Seedling expression: High GFP expression invasculature of hypocotyls, cotyledons and root. GFP expression inepidermis and mesophyll cells of cotyledons. T2 Mature expression: HighGFP expression in vasculature and guard cells of sepals and in anthers,petals and silique of flowers. GFP expressed in style, carpels andvasculature of silique. Not expressed in ovules or seed. Low GFPexpression in leaf vasculature. High GFP expression in root. SourcePromoter Organism: Arabidopsis thaliana, Columbia (Col) ecotype Vector:pNewbin4-HAP1-GFP Marker Type: GFP-ER Generation Screened: X T1 Mature XT2 Seedling X T2 Mature Inductions completed. Events Screened/Treatment: Age: Gen: Time points: Response Response: 1. ABA—[uM] 14 daysT2  4 hrs 6/0 No 2. Heat—28 C. 15 days T2  24 hrs 3/3 Low 3. Heat—36 C. 9 days T2 >24 hrs 6/3 High 4. Heat—41 C. 14 days T2  4 hrs 3/3 Medium5. Heat—41 C. 15 days T2  24 hrs 3/0 No 6. Heat—46 C. 14 days T2  4 hrs3/0 No 7. Heat—40 C.  4 wks T2  24 hrs 6/6 Low 8. Drought  4 wks T2 1.0%moisture 6/6 High Inducible expression summary: Treatment: Time pointinduced: Organs induced: Tissues induced: 2. Heat—28 C.  24 hrsCotyledons Rosette leaf 3. Heat—36 C. >24 hrs Cotyledons Ep, Me, VsRosette leaf Ep, Me, Vs Root Vs 4. Heat—41 C.  4 hrs Cotyledons Rosetteleaf 7. Heat—40 C.  24 hrs Flower Se, Pe, Vs Stem Ph, Vs Leaf Vs 8.Drought 1.0% moisture Flower Se, Pe, Ca Stem Xy, Ph, Vs Leaf Ep, Me RootEp

TABLE 1-14 T1 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 6 Events Expressing: n = 1 (04) Expression DetectedInflorescence H flowers

TABLE 2-14 T2 Seedling Expression Tissues Screened Events Screened: n =6 Events Expressing: n = 2 (04, 05) Expression Detected Hypocotyl Hvascular Cotyledon H mesophyll H vascular H epidermis Primary Root Hvascular

TABLE 3-14 T2 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 6 Events Expressing: n = 6 (01-06) Expression DetectedFlower H sepal H petal H anther H vascular H stomata H silique Silique Hstyle H carpel H funiculus H vascular Leaf L vascular Aerial organs Hinflorescence Root H mature root

TABLE 5-14 Promoter utility Trait Area: Water Use Efficiency Sub-traitArea: Drought Tolerance, Heat Stress Tolerance Utility: Among other usesthis promoter sequence is useful to improve plant tolerance to heat anddrought stress.

Construct: YP2571 Promoter candidate I.D: 29223786 cDNA I.D: 23618816Events expressing: 01-06

Promoter Expression Report YP2590 (SEQ ID NO: 15) Promoter Tested In:Arabidopsis thaliana, Wassilewskija (WS) ecotype Spatial expressionsummary: Arabidopsis Flower H pedicel Silique H ovule OvulePre-fertilization: H funiculus Post-fertilization: H funiculus Aerialorgans H flowers Root H mature root Rice Root H not-specific Meristem Hnot-specific Observed expression pattern: Arabidopsis: T1 Matureexpression: High GFP expression at the distal end of the funiculus indeveloping ovules and seed. GFP highly localized to adaxial side at thebase of the pedicel, in structures resembling stipules of leaves. T2Seedling expression: No GFP expression detected. T2 Mature expression:GFP detected at the base of pedicles and roots. Rice: T0 Seedlingexpression: GFP expression was detected strongly throughout the root andin meristematic tissues corresponding to the stem nodes. Source PromoterOrganism: Arabidopsis thaliana, Columbia (Col) ecotype Vector:pNewbin4-HAP1-GFP Marker Type: GFP-ER Generation Screened: X T1 Mature XT2 Seedling X T2 Mature

TABLE 1-14 T1 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 6 Events Expressing: n = 3 Expression Detected Flower Hpedicel Silique H ovule Ovule Pre-fertilization: H funiculusPost-fertilization: H funiculus

TABLE 2-14 T2 Seedling Expression Tissues Screened Events Screened: n =6 Events Expressing: n = 0 No GFP Expression Detected

TABLE 3-14 T2 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 6 Events Expressing: n = 2 (01, 03, 04, 06) ExpressionDetected Aerial organs H flowers Root H mature root

TABLE 4-14 T0 Rice Seedlings Organs/Tissues screened Events Screened: n= 13 Events Expressing: n = 4 (01, 02, 06, 09) Expression Detected RootH not-specific Meristem L not-specific

TABLE 5-14 Promoter utility Trait Area: Yield, PG&D, Confinement, WaterUse Efficiency Sub-trait Area: Seed Number, Seed Growth, Plant Size,Growth Rate Utility: Among other uses this promoter sequence is usefulto improve plant architecture and increase the number of secondaryfloral branches. Expression in the root could be valuable for improveduptake and transport of water and nutrients.

Arabidopsis Construct: YP2590 -Arabidopsis Promoter candidate I.D:25659363 cDNA I.D: 23523207 Events expressing: 02, 03, 04 RiceConstruct: PD3146 Promoter candidate I.D: 55210297 cDNA I.D: 23523207Events expressing: 01, 02, 06, 09

Promoter Expression Report YP2606 (SEQ ID NO: 16) Promoter Tested In:Arabidopsis thaliana, Wassilewskija (WS) ecotype Spatial expressionsummary: Primary Root H epidermis H cortex Mature Root H epidermis Lcortex H stele H vasculature Observed expression pattern: T1 Matureexpression: No GFP expression detected. T2 Seedling expression: Rootspecific GFP expression. GFP expressed in root epidermis and cortexcells. T2 Mature expression: Root specific GFP expression. GFP expressedin root epidermis and the stele, non-ground cell vascular region ofroot. Source Promoter Organism: Arabidopsis thaliana, Columbia (Col)ecotype Vector: pNewbin4-HAP1-GFP Marker Type: GFP-ER GenerationScreened: X T1 Mature X T2 Seedling X T2 Mature

TABLE 1-15 T1 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 6 Events Expressing: n = 0 No Expression Detected

TABLE 2-15 T2 Seedling Expression Tissues Screened Events Screened: n =6 Events Expressing: n = 3 (04-06) Expression Detected Primary Root HepidermisH cortex

TABLE 3-15 T2 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 6 Events Expressing: n = 2 (04, 06) Expression DetectedMature Root H epidermis H cortex H stele H vasculature

TABLE 4-15 Promoter utility Trait Area: Water Use Efficiency, NutrientUse Efficiency Sub-trait Area: Drought Tolerance, Nitrogen andPhosphorous Utilization Utility: Among other uses this promoter sequenceis useful to improve the uptake of water and nutrients.

Construct: YP2606 Promoter candidate I.D: 25086987 cDNA I.D: 36511228Events expressing: 04-06One or more fragments of the above described promoter are identified inthe miscellaneous feature section of the relevant SEQ ID in the SequenceListing. Those fragments were tested for promoter activity by the sameprocedures as described above, and the results are summarized below.Ceres Promoter PD3464 expresses weakly in roots in rice.

Promoter Expression Report YP2608 (SEQ ID NO: 17) Promoter Tested In:Arabidopsis thaliana, Wassilewskija (WS) ecotype Spatial expressionsummary: Primary Root H epidermis H cortex Aerial organs H flowersFlower H stamen H filament H anther Mature Root H epidermis L cortex Hstele Observed expression pattern: T1 Mature expression: No GFPexpression detected. T2 Seedling expression: GFP expressed in rootepidermis. T2 Mature expression: High stamen specific GFP expressioncorresponding to third organ whorl in Arabidopsis flower. GFP expressedin sub-epidermal cells in root. Source Promoter Organism: Arabidopsisthaliana, Columbia (Col) ecotype Vector: pNewbin4-HAP1-GFP Marker Type:GFP-ER Generation Screened: X T1 Mature X T2 Seedling X T2 Mature T3Seedling

TABLE 1-16 T1 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 4 Events Expressing: n = 0 No GFP Expression Detected

TABLE 2-16 T2 Seedling Expression Tissues Screened Events Screened: n =5 Events Expressing: n = 5 (01-05) Expression Detected Primary Root Hepidermis H cortex

TABLE 3-16 T2 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 4 Events Expressing: n = 3 (02, 03, 04) ExpressionDetected Flower H stamen H filament H anther Aerial organs H flowersMature Root H epidermis L cortex H stele

TABLE 4-16 Promoter utility Trait Area: Water Use Efficiency, NutrientUse Efficiency, Sterility Sub-trait Area: Drought Tolerance, Nitrogenand Phosphorous Utilization, Male Sterility Utility: Among other usesthis promoter sequence is useful to improve the uptake of water andnutrients from the soil. It could also be used to engineer malesterility.

Construct: YP2608 Promoter candidate I.D: 25656951 cDNA I.D:  5680676Events expressing: 02, 03 ,04One or more fragments of the above described promoter are identified inthe miscellaneous feature section of the relevant SEQ ID in the SequenceListing. Those fragments were tested for promoter activity by the sameprocedures as described above, and the results are summarized below. ForCeres Promoter PD3739, no expression is observed.

Promoter Expression Report YP2683 (SEQ ID NO: 18) Promoter Tested In:Arabidopsis thaliana, Wassilewskija (WS) ecotype Spatial expressionsummary: Flower L petal L anther H pollen Stem L cortex L vascular Hxylem H procambium Leaf H vascular L epidermis L stomata Cotyledon Lepidermis L hydathode Primary Root L cortex L epidermis H vascularAerial Organs L flower Mature Root L epidermis H cortex H vascular Hquiescent center Observed expression pattern: T1 Mature expression:Expression is primarily in pollen and in vascular tissues in leaves andstems. Expression appears to be strongest in the meristematic vascularcells (procambium), which gives rise to the xylem and phloem. T2Seedling expression: Expression is observed primarily in the rootvasculature. Weak expression was also observed in the epidermis of theseedling. T2 Mature: Weak expression was observed in the floral tissue.Strong expression was detected in the root, primarily in the cortex andvascular tissues as well as the quiescent center. Source PromoterOrganism: Arabidopsis thaliana, Columbia (Col) ecotype Vector:pNewbin4-HAP1-GFP Marker Type: GFP-ER Generation Screened: X T1 Mature XT2 Seedling X T2 Mature

TABLE 1-17 T1 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 8 Events Expressing: n = 3 (02, 03, 06) ExpressionDetected Flower L petal L anther H pollen Stem L cortex L vascular Hxylem H procambium Leaf H vascular L epidermis L stomata

TABLE 2-17 T2 Seedling Expression Tissues Screened Events Screened: n =6 Events Expressing: n = 3 (02, 05, 06) Expression Detected Cotyledon Lepidermis L hydathode Primary Root L epidermis L cortex H vascular

TABLE 3-17 T2 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 6 Events Expressing: n = 4 (02, 03, 05, 06) ExpressionDetected Aerial organs L flower Mature Root L epidermis H cortex Hvascular H quiescent center

TABLE 4-17 Promoter utility Trait Area: Sterility, Water Use Efficiency,Nutrient Use Efficiency Sub-trait Area: Male-Sterility, DroughtTolerance, Nitrogen and Phosphorous Utilization Utility: Among otheruses this promoter sequence is useful to improve the uptake of water andnutrients from the soil. The pollen expression could be used to engineermale sterility for gene-confinement.

Construct: YP2683 Promoter candidate I.D: 41958148 cDNA I.D: 36558613Events expressing: 02, 03, 05, 06

Promoter Expression Report YP2816 (SEQ ID NO: 19) Promoter Tested In:Arabidopsis thaliana, Wassilewskija (WS) ecotype Spatial expressionsummary: Flower H vascular H ovule Silique H septum OvulePre-fertilization: H outer integument Post-fertilization: H seed coat Hembryo Embryo H radicle H late mature Stem H epidermis H vascular Hxylem H phloem Leaf L vascular H trichome Hypocotyl L epidermisCotyledon L epidermis Rosette Leaf H epidermis H trichome H primordiaPrimary Root H epidermis H cortex H vascular L quiescent center Lcolumella Mature Root H epidermis H cortex H vascular H endodermis Hparenchyma H stele Observed expression pattern: All cells in root. T1Mature expression: Promoter expression is observed in the pre-fertilized ovule integuments as well as the seed coat and the developingradicle of the embryo. Expression is also observed weakly in thevasculature of the stem and leaf trichomes. T2 Seedling expression:Seedling expression is primarily in all cells of the root. T2 Mature:Expression is strong in all cells of the root analyzed. Source PromoterOrganism: Arabidopsis thaliana, Columbia (Col) ecotype Vector:pNewbin4-HAP1-GFP Marker Type: GFP-ER Generation Screened: X T1 Mature XT2 Seedling X T2 Mature

TABLE 1-18 T1 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 8 Events Expressing: n = 5 (02, 03, 05, 07, 08) ExpressionDetected Flower H vascular H ovule Silique H septum OvulePre-fertilization: H outer integument Post-fertilization: H seed coat Hembryo Embryo H late H radicle Stem H epidermis H vascular H xylem Hphloem Leaf L vascular H trichome

TABLE 2-18 T2 Seedling Expression Tissues Screened Events Screened: n =6 Events Expressing: n = 3 (02, 03, 05) Expression Detected Hypocotyl Lepidermis Cotyledon L epidermis Rosette Leaf H epidermis H trichome Hprimordia Primary Root H epidermis H cortex H vascular L quiescentcenter L columella

TABLE 3-18 T2 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 6 Events Expressing: n = 5 (01-05) Expression DetectedLeaf H trichome Aerial organs L stem Mature Root H epidermis H cortex Hvascular H endodermis H parenchyma H stele

TABLE 4-18 Promoter utility Trait Area: Water Use Efficiency, NutrientUse Efficiency Sub-trait Area: Drought Tolerance, Nitrogen andPhosphorous Use Efficiency Utility: Among other uses this promotersequence is useful to improve the uptake and utilization of water andnutrients from the soil and the transport of water throughout thevasculature.

Construct: YP2816 Promoter candidate I.D: 15372106 cDNA I.D: 36540030Events expressing: 01-05, 07, 08One or more fragments of the above described promoter are identified inthe miscellaneous feature section of the relevant SEQ ID in the SequenceListing. Those fragments were tested for promoter activity by the sameprocedures as described above, and the results are summarized below.Ceres Promoter PD3238 expresses weakly in roots in rice. No expressionis observed for Ceres Promoter PD3229 and Ceres Promoter PD3243.

Promoter Expression Report YP2832 (SEQ ID NO: 20) Promoter Tested In:Arabidopsis thaliana, Wassilewskija (WS) ecotype Spatial expressionsummary: Flower H pedicel H receptacle H style Silique H style H septumH abscission zone Stem H epidermis H cortex Leaf L stomata Primary RootL epidermis H cortex H endodermis H phloem Lateral Root H primordiaAerial Organs H stem Mature Root L epidermis H cortex H endodermis Hphloem H lateral roots Observed expression pattern: T1 Matureexpression: Expression is primarily observed in the epidermis andcortical layers of the stem tissue. T2 Seedling expression: The promoteris expressed in the most mature parts of the primary root, and is notdetected in the root tips. The root expression is also observed in thebuds giving rise to the secondary root branches. No expression isobserved in the hypocotyl or cotyledons. T2 Mature expression:Expression observed in non-leaf tissues. Source Promoter Organism:Arabidopsis thaliana, Columbia (Col) ecotype Vector: pNewbin4-HAP1-GFPMarker Type: GFP-ER Generation Screened: X T1 Mature X T2 Seedling XT2Mature

TABLE 1-19 T1 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 6 Events Expressing: n = 5 (01-05) Expression DetectedFlower H pedicel H receptacle H style Silique H style H septum Habscission zone Stem H epidermis H cortex Leaf L stomata

TABLE 2-19 T2 Seedling Expression Tissues Screened Events Screened: n =6 Events Expressing: n = 4 (01, 02, 04, 05) Expression Detected PrimaryRoot L epidermis H cortex H endodermis H phloem Lateral root H primordia

TABLE 3-19 T2 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 6 Events Expressing: n = 6 (01-06) Expression DetectedAerial organs H stem Mature Root H lateral root L epidermis H cortex Hendodermis H phloem

TABLE 4-19 Promoter utility Trait Area: Source, Water Use Efficiency,Nutrient Use Efficiency Sub-trait Area: Carbon/Nitrogen Partitioning,Drought Tolerance, Nitrogen and Phosphorous Use Efficiency Utility:Among other uses this promoter sequence is useful to engineer thestorage of carbon into stem and root material, to improve the uptake ofwater and nutrients into the roots from the soil, and to protect theplants from pests.

Construct: YP2832 Promoter candidate I.D: 32258957 cDNA I.D: 36551046Events expressing: 01-06

Promoter Expression Report PD2995 (aka PD2263) (SEQ ID NO: 21) PromoterTested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype Spatialexpression summary: Flower H epidermis L petal H carpel H style Hsilique H pedicel Silique H epidermis H style H carpel H septum H ovuleOvule Pre-fertilization: H outer integument Post-fertilization: H seedcoat H outer integument Embryo H mature endosperm H mature Stem Hepidermis H pith Leaf L epidermis H mesophyll Inflorescence H floralmeristem H floral primordia Hypocotyl H epidermis H cortex H vascularCotyledon H epidermis H mesophyll Rosette Leaf H epidermis H mesophyll Hstipule Primary Root H epidermis H cortex H vascular H quiescent centerH root meristem H root cap Lateral root H primordia H flanking cells Hlateral root cap Aerial organs H inflorescence H floral meristem Hfloral primordia H flowers H silique H stem H leaf Root H mature rootObserved expression pattern: T1 Mature expression: High broad GFPexpression throughout aerial organs. High GFP expression in flowers,stems, and leaves. GFP expressed in floral meristems and primordia. HighGFP expression in carpels, ovules and developing seed in silique. GFPexpressed in outer integuments, endosperm, seed coat and embryos inovules. GFP expressed in pith and epidermis in stem and epidermis andmesophyll of leaf. T2 Seedling expression: High GFP expression in aerialorgans and root. High GFP expression in epidermis and mesophyll cells ofcotyledon and rosette leaf. High GFP expression in epidermis, cortex andvasculature of hypocotyl and root. GFP expressed in root meristem cellsand root cap. T2 Mature expression: High broad GFP expression throughoutaerial organs and roots. Source Promoter Organism: Arabidopsis thaliana,Columbia (Col) ecotype Vector: CRS338-GFP Marker Type: GFP-ER GenerationScreened: X T1 Mature X T2 Seedling X T2 Mature

TABLE 1-20 T1 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 6 Events Expressing: n = 6 (01-06) Expression Detected XFlower H pedicel L petal H carpel H style H epidermis H silique XSilique H style H carpel H septum H epidermis H ovule X OvulePre-fertilization: H outer integument Post-fertilization: H outerintegument H seed coat H mature endosperm X Embryo H mature X Stem Hepidermis H pith X Leaf H mesophyll L epidermis X Inflorescence H floralmeristem H floral primordia

TABLE 2-20 T2 Seedling Expression Tissues Screened Events Screened: n =6 Events Expressing: n = 6 (01-06) GFP Expression Detected X Hypocotyl Hepidermis H cortex H vascular X Cotyledon H mesophyll H epidermis XRosette Leaf H mesophyll H epidermis H stipule X Primary Root Hepidermis H cortex H vascular H quiescent center H root meristem H rootcap X Lateral root H primordia H flanking cells □ H lateral root cap

TABLE 3-20 T2 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 6 Events Expressing: n = 6 (01-06) GFP Expression DetectedX Aerial organs H inflorescence H floral meristem H floral primordia Hflowers H silique H stem H leaf X Root H mature root

TABLE 4-20 Promoter utility Trait Area: Water Use Efficiency, PG&D,Seed, Nutrient, and Yield Sub-trait Area: Water use efficiency, growthrate, seed yield, and nutrient utilization Utility: Among other usesthis promoter sequence is useful to modulate plant growth andarchitecture and the utilization and water and nutrients.

Construct: PD2263 Promoter candidate I.D: 38960222 cDNA I.D: 36549595Events expressing: 01-06One or more fragments of the above described promoter are identified inthe miscellaneous feature section of the relevant SEQ ID in the SequenceListing. Those fragments were tested for promoter activity by the sameprocedures as described above, and the results are summarized below.

For Ceres Promoter PD2926, expression is weak compared to thefull-length promoter PD2995; the promoter remains active in all tissuesexcept the embryo.

For Ceres Promoter PD3048, expression is weak compared to thefull-length promoter PD2995; the promoter remains active in all tissues.

For Ceres Promoter PD3182, no expression is observed.

For Ceres Promoter PD3345, expression is very weak.

For Ceres Promoter PD3503, no expression is observed.

For Ceres Promoter PD3676, expression is weak compared to thefull-length promoter PD2995; the promoter expresses at higher levels invegetative tissues than in reproductive tissues.

Promoter Expression Report PD2999 (aka PD2258) (SEQ ID NO: 22) PromoterTested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype Flower Hpedicel H sepal H carpel H style H stigma H silique Silique H stigma Hstyle H carpel H placentae H funiculus H epidermis H ovule OvulePre-fertilization: H primordia H inner integument H outer integument Hfuniculus Post-fertilization: H outer integument H seed coat H earlyendosperm H mature endosperm H embryo Embryo H suspensor H heart Hmature H radicle H cotyledons Stem H epidermis H cortex Hinterfascicular region H vascular H xylem H phloem H pith Leaf Hmesophyll H epidermis Inflorescence H floral meristem H floral primordiaHypocotyl H epidermis H cortex H vascular Cotyledon H epidermis Hvascular Rosette Leaf H epidermis Primary Root H vascular H root capObserved expression pattern: T1 Mature expression: Broad high GFPexpression throughout aerial organs. High GFP expression ininflorescence, floral meristem, flowers, siliques. High GFP expressionthroughout tissues of silique. GFP expressed in carpels, placenta, ovuleprimordia, developing ovules, embryo, and endosperm. High GFP expressionin outer integuments and seed coats of developing ovules and seed. HighGFP expression in vascular and ground tissues of stem. High GFPexpression in epidermis and mesophyll cells of leaf. T2 Seedlingexpression: Broad expression throughout aerial organs and vasculature ofroot. Source Promoter Organism: Arabidopsis thaliana, Columbia (Col)ecotype Vector: CRS338-GFP Marker Type: GFP-ER Generation Screened: X T1Mature X T2 Seedling

TABLE 1-21 T1 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 6 Events Expressing: n = 4 (01, 02, 04, 06) GFP ExpressionDetected X Flower H pedicel H sepal H carpel H style H stigma H siliqueX Silique H stigma H style H carpel H placentae H funiculus H epidermisH ovule X Ovule Pre-fertilization: H primordia H inner integument Houter integument H funiculus Post-fertilization: H outer integument Hseed coat H early endosperm H mature endosperm H embryo X Embryo Hsuspensor H heart H mature H radicle H cotyledons X Stem H epidermis Hcortex H interfascicular region H vascular H xylem H phloem H pith XLeaf H mesophyll H epidermis X Inflorescence H floral meristem H floralprimordia

TABLE 2-21 T2 Seedline Expression Tissues Screened Events Screened: n =6 Events Expressing: n = 5 (01-04, 06) □ No GFP Expression Detected XHypocotyl H epidermis H cortex H vascular X Cotyledon H epidermis Hvascular X Rosette Leaf H epidermis X Primary Root H vascular H root cap

TABLE 3-21 Promoter utility Trait Area: Water use efficiency, PG&D,Seed, Nutrient, Yield Sub-trait Area: Water use efficiency, growth rate,seed size and yield, and nutrient use Utility: Among other uses thispromoter sequence is useful to improve: Water use efficiency, PG&D,Seed, Nutrient, Yield

Construct: PD2258 Promoter candidate I.D: 38960200 cDNA I.D: 23478038Events expressing: 01, 02, 04, 06One or more fragments of the above described promoter are identified inthe miscellaneous feature section of the relevant SEQ ID in the SequenceListing. Those fragments were tested for promoter activity by the sameprocedures as described above, and the results are summarized below.

For Ceres Promoter PD2929, expression is very weak compared to thefull-length promoter PD2999; the promoter remains active in all tissues.

For Ceres Promoter PD3183, expression is only detected in anthers andstigma.

For Ceres Promoter PD3240, expression is very weak compared to thefull-length promoter PD2999; the promoter remains active in all tissues.

For Ceres Promoter PD3266, no expression is detected in rice.

Promoter Expression Report PD3141 (SEQ ID NO: 23) Promoter Tested In:Oryza sativa Spatial expression summary: Tiller: not-specific Main culm:bundle sheath, endodermis, epidermis, internode, ligule, node,pericycle, phloem, sclerenchyma layer, vasculature, xylem Root: cortex,epidermis, vascular Leaf: epidermis, leaf blade, leaf sheath, mesophyll,petiole, stipule, stomata, vasculature Meristem: floral meristem, shootapical meristem, vegetative meristem Panicle: flag leaf, ovary,peduncle, primary branch, rachilla, rachis, spiklet Spikelet: floralmeristem, shoot apical meristem, vegetative meristem Observed expressionpattern: Broad Source Promoter Organism: Oryza sativa Vector: Binary DFEGFP Marker Type: EGFP Generation Screened: T0 Seedling and T0 MatureInduction Data

TABLE 1-22 T0 Seedling Expression Organs/Tissues screened EventsScreened: n = 12 Events Expressing: n = 9 (01, 03-04, 06-11) OrgansTiller: not-specific Main culm: not-specific Root: not-specific Leaf:not-specific Meristem: not-specific

TABLE 2-22 T0 Mature Plant Expression Tissues Screened Events Screened:n = 6 Events Expressing: n = 6 (01, 04, 07-08, 11, 14) Organs Main culm:bundle sheath, endodermis, epidermis, internode, ligule, node,pericycle, phloem, sclerenchyma layer, vasculature, xylem Root: cortex,vascular Panicle: flag leaf, ovary, peduncle, primary branch, rachilla,rachis, spiklet Spiklet: flag leaf, floret(palea), lemma, ovule,pedicle, pollen, seed, stigma Leaf: epidermis, leaf blade, leaf sheath,mesophyll Meristem: floral meristem, shoot apical meristem, vegetativemeristem Promoter utility Trait Area: Yield, Composition, Disease,Stress Tolerance, Nutrient Use Efficiency, Nutrient UtilizationSub-trait Area: Biomass, Lignin composition, Disease resistance, Salttolerance, Drought tolerance, Phosphate and Nitrate Use Efficiency,Phosphate and Nitrate Utilization Utility: Among other uses, thispromoter sequence is useful to improve: the biomass of the plants undernormal and stressful conditions through the overexpression oftrait-specific transgenes.

Construct: PD3141 SR/OS Line: OS00486 Promoter candidate I.D: 54507599cDNA I.D: NA Events expressing: 01, 03-04, 06-11, 14

Promoter Expression Report YP2680 (SEQ ID NO: 24) Promoter Tested In:Arabidopsis thaliana Spatial expression summary: Leaf: epidermis,vasculature Flower: vascular Silique: abscission zone, vascular Primaryroot: cortex, epidermis, root hairs, vascular Lateral root: cortex,epidermis, vascular Mature root: not-specific Observed expressionpattern: Primarily root expression Source Promoter Organism: Arabidopsisthaliana Vector: Binary TC(815) Marker Type: erGFP Generation Screened:T1 Mature, T2 Seedling, T2 Mature

TABLE 1-23 T1 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 8 Events Expressing: n = 7 (01, 03-08) Organs Leaf:epidermis, vasculature Flower: vascular Silique: abscission zone,vascular

TABLE 2-23 T1 Seedling Expression Tissues Screened Events Screened: n =5 Events Expressing: n = 5 (01-05) Primary root: cortex, epidermis, roothairs, vascular Lateral root: cortex, epidermis, vascular

TABLE 3-23 T2 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 6 Events Expressing: n = 5 (01-03, 06, 08) Organs Matureroot: not-specific Primary root: cortex, epidermis, vascular Lateralroot: cortex, epidermis, vascular Promoter utility: Trait Area: DroughtTolerance, Nutrient Utilization Sub-trait Area: Water Utilization,Phosphate and Nitrate Utilization Utility: Among other uses, thispromoter sequence is useful to improve: the uptake and transport ofwater and nutrients from the soil to support vegetative growth. Notes:1000 nt upstream of atg

Construct: YP2664 SR/OS Line: SR04406 Promoter candidate I.D: 41958160cDNA I.D: 36511557 Events expressing: 01-08One or more fragments of the above described promoter are identified inthe miscellaneous feature section of the relevant SEQ ID in the SequenceListing. Those fragments were tested for promoter activity by the sameprocedures as described above, and the results are summarized below.Ceres Promoter PD3584 is a strong, broadly expressing promoter;comparable to the full-length promoter PD3141.

Promoter Expression Report PD3147 (aka YP2663) (SEQ ID NO: 25) PromoterTested In: Arabidopsis thaliana, Wassilewskija (WS) ecotype Spatialexpression summary: Flower H pedicel H sepal H petal H filament H antherL carpel L silique Silique L carpel L funiculus Stem H epidermis Hcortex H vascular H xylem H phloem L pith Leaf H epidermis H mesophyll Hvascular Hypocotyl H epidermis H cortex Cotyledon H epidermis Hmesophyll Rosette Leaf H epidermis H mesophyll Aerial organs Hinflorescence H flowers L silique H stem H leaf Observed expressionpattern: T1 Mature expression: High GFP expressed in aerial tissues.High GFP expression in leaf, stem and inflorescence. High GFP expressedin pedicels, sepals, petals and stamens. Low GFP expression in epidermisof silique. No GFP expression observed in embryo. High GFP expression inepidermis, cortex, phloem and xylem cells of vasculature. Low GFPexpression in pith sells of stem. High GFP expression in epidermis,mesophyll and vasculature of leaves. T2 Seedling expression: High GFPexpressed in aerial tissues. High GFP expression in epidermis andmesophyll cells of cotyledons and rosette leaves and in epidermis andcortex of hypocotyls. No GFP expression observed in roots. T2 Matureexpression: GFP expressed in aerial tissues. High GFP expression inleaf, stem and inflorescence. No GFP expression observed in roots.Source Promoter Organism: Arabidopsis thaliana, Columbia (Col) ecotypeVector: pNewbin4-HAP1-GFP Marker Type: GFP-ER Generation Screened: X T1Mature X T2 Seedling X T2 Mature Inductions completed. Events Screened/Treatment: Age: Gen: Time points: Response Response: 1. Nitrogen—high Nto low N 4 wks T2 72 hrs 6/0 No [14.3 mM KNO₃ to 28.6 mM Mannitol]Inducible expression summary: Treatment: Time point induced: Organsinduced: Tissues induced:

TABLE 1-24 T1 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 6 Events Expressing: n = 4 (02, 04-06) GFP ExpressionDetected X Flower H pedicel H sepal H petal H filament H anther L carpelL silique X Silique L carpel L funiculus X Stem H epidermis H cortex Hvascular H xylem H phloem L pith X Leaf H mesophyll H vascular Hepidermis

TABLE 2-24 T2 Seedling Expression Tissues Screened Events Screened: n =6 Events Expressing: n = 4 (02, 04-06) GFP Expression Detected XHypocotyl H epidermis H cortex X Cotyledon H mesophyll H epidermis XRosette Leaf H mesophyll H epidermis

TABLE 3-24 T2 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 5 Events Expressing: n = 4 (02, 04-05) GFP ExpressionDetected X Aerial organs H inflorescence H flowers L silique H stem Hleaf

TABLE 5-24 Promoter utility Trait Area: PG&D, Confinement, HybridProduction Sub-trait Area: Plant Size, Growth Rate, Carbon FixationUtility: Among other uses this promoter sequence is useful to improvecarbon fixation in above ground tissues to increase biomass and seedyield. Expression is useful for engineering of carbon and nitrogenratios. Expression in flowers is deployed for sterility and confinement.

Construct: YP2663 Promoter candidate I.D: 25087104 cDNA I.D: 36567145Events expressing: 02, 04-06One or more fragments of the above described promoter are identified inthe miscellaneous feature section of the relevant SEQ ID in the SequenceListing. Those fragments were tested for promoter activity by the sameprocedures as described above, and the results are summarized below.Ceres Promoter PD3721 is a strong, vegetatively expressing promoter;comparable to the full-length promoter PD3147.

Promoter Expression Report For PT0822 (SEQ ID NO: 26) Promoter TestedIn: Arabidopsis thaliana, Wassilewskija (WS) ecotype Observed expressionpattern: T1 Mature expression: Expression observed in mature root only.T2 Seedling expression: Expression specific to epidermis and cortexcells of root. Source Promoter Organism: Arabidopsis thaliana, Columbia(Col) ecotype Vector: pNewbin4-HAP1-GFP Marker Type: GenerationScreened: T1 Mature T2 Seedling T2 Mature

TABLE 1-25 T1 Mature Plant Expression Organs/Tissues screened Root Hepidermis

TABLE 2-25 T2 Seedling Expression Tissues Screened Expression DetectedPrimary Root H epidermis H cortex

Promoter Expression Report for PD3389 (nucleotides 601-1000 of SEQ IDNO: 26) Promoter Tested In: Oryza sativa Spatial expression summary: T0Seedling X Root expression in: CORTEX EPIDERMIS ROOT CAP T0 Mature XRoot expression: non-specific. Observed expression pattern: T0 Seedling:Expression observed in the root only. T2 Seedling expression: Expressionspecific to epidermis and cortex cells of root. Deletion ofroot-specific promoter PT0822. 600 nucleotides were deleted from the5′end of the 1000 nucleotide PT0822 promoter (SEQ ID NO: 26). SourcePromoter Organism: Arabidopsis thaliana, Columbia (Col) ecotypeGeneration Screened: T0 Seedling and T0 Mature

TABLE 1-26 T0 Seedling Expression Organs/Tissues screened EventsScreened: n = 16 Events Expressing: n = 3 (01, 05,13) Organs X Rootexpression in: CORTEX EPIDERMIS ROOT CAP

TABLE 2-26 T0 Mature Plant Expression Organs/Tissues screened EventsScreened: n = 4 Events Expressing: n = 2 (05,13) Organs X Rootexpression: nonspecific

Promoter utility Trait Area: Salt tolerance, Drought Tolerance, NutrientUse Efficiency, Nutrient Utilization Sub-trait Area: Salt tolerance,Drought tolerance, Phosphate and Nitrate Use Efficiency, Phosphate andNitrate Utilization Utility: Among other uses, this promoter sequence isuseful to improve: the biomass of the plants under normal and stressfulconditions through the overexpression of trait-specific transgenes.One or more fragments of the above described promoter are identified inthe miscellaneous feature section of the relevant SEQ ID in the SequenceListing. Those fragments were tested for promoter activity by the sameprocedures as described above, and the results are summarized below. ForCeres Promoter PD3389, expression is observed in the roots of riceseedlings.

The invention being thus described, it will be apparent to one ofordinary skill in the art that various modifications of the materialsand methods for practicing the invention can be made. Such modificationsare to be considered within the scope of the invention as defined by thefollowing claims.

Each of the references from the patent and periodical literature citedherein is hereby expressly incorporated in its entirety by suchcitation.

1. An isolated nucleic acid molecule having promoter activity comprisinga nucleotide sequence selected from the group consisting of: a) anucleotide sequence according to any one of SEQ ID NOs. 1-26; b) anucleotide sequence of nucleic acid residues 601-1000 of SEQ ID NO: 26;c) a nucleotide sequence comprising a functional fragment of (a) or (b),wherein said fragment has promoter activity, and wherein said isolatednucleic acid molecule is not SEQ ID NO:
 5. 2. An isolated nucleic acidmolecule comprising a nucleotide sequence that shows at least 80 percentsequence identity to any one of SEQ ID NOs. 1-26 or nucleic acidresidues 601-1000 of SEQ ID NO: 26, wherein said nucleic acid moleculecomprises a regulatory region that directs transcription of an operablylinked heterologous polynucleotide, and wherein said isolated nucleicacid molecule is not SEQ ID NO:
 5. 3. The isolated nucleic acid moleculeof claim 2, wherein said nucleotide sequence shows at least 85 percentsequence identity to any one of SEQ ID NOs: 1-26 or nucleic acidresidues 601-1000 of SEQ ID NO:
 26. 4. The isolated nucleic acidmolecule of claim 2, wherein said nucleotide sequence has at least 90percent sequence identity to any one of SEQ ID NOs: 1-26 or nucleic acidresidues 601-1000 of SEQ ID NO:
 26. 5. The isolated nucleic acidmolecule according to claim 1, wherein said regulatory region comprisesat least one member selected from the group consisting of a promoter, anenhancer and an intron.
 6. An isolated nucleic acid molecule consistingof the nucleotide sequence according to any one of SEQ ID Nos. 1-4, 6-26and the nucleic acid residues 601-1000 of SEQ ID NO:
 26. 7. A vectorconstruct comprising: a) a first nucleic acid molecule according toclaim 1; and b) a transcribable polynucleotide molecule, wherein saidfirst nucleic acid molecule and said transcribable polynucleotidemolecule are heterologous to each other and are operably linked.
 8. Thevector construct according to claim 7, wherein said first nucleic acidmolecule consists of the nucleic acid molecule set forth in any one ofSEQ ID NOs: 1-26 or nucleic acid residues 601-1000 of SEQ ID NO:
 26. 9.The vector construct according to claim 7, wherein said transcribablepolynucleotide molecule encodes a polypeptide.
 10. The vector constructaccording to claim 9, wherein said transcribable polynucleotide moleculeis operably linked to said first nucleic acid molecule in the senseorientation.
 11. The vector construct according to claim 10, whereinsaid transcribable polynucleotide molecule is transcribed into an RNAmolecule that expresses the polypeptide encoded by transcribablepolynucleotide molecule.
 12. The vector construct according to claim 7,wherein said transcribable polynucleotide second nucleic acid moleculeis operably linked to said first nucleic acid molecule in the antisenseorientation.
 13. The vector construct according to claim 12, whereinsaid transcribable polynucleotide molecule is transcribed into anantisense RNA molecule.
 14. The vector construct according to claim 7,wherein said transcribable polynucleotide molecule is transcribed intoan interfering RNA against an endogenous gene.
 15. The vector constructaccording to claim 9, wherein said transcribable polynucleotide moleculeencodes a polypeptide of agronomic interest.
 16. A plant or plant cellcomprising: a) the nucleic acid molecule according to claim 1 that isoperably linked to a heterologous polynucleotide, or b) the vectorconstruct according to claim
 7. 17. A plant or plant cell stablytransformed with the vector construct according to claim
 7. 18. A seedof a plant according to claim
 16. 19. A method of directingtranscription by combining, in an environment suitable fortranscription: a) a first nucleic acid molecule according to any claim1; and b) a transcribable polynucleotide molecule; wherein said firstnucleic acid molecule and said transcribable polynucleotide molecule areheterologous to each other and operably linked.
 20. A method ofexpressing an exogenous coding region in a plant comprising: (a)transforming a plant cell with the vector of claim 7; (b) regenerating astably transformed plant from the transformed plant cell of step (a);and (c) selecting plants containing a transformed plant cell, whereinexpression of the transcribable polynucleotide molecule results inproduction of a polypeptide encoded by said transcribable polynucleotidemolecule.
 21. A method of altering the expression of a gene in a plantcomprising: a) transforming a plant cell with the nucleic acid moleculeaccording to claim 1 that is operably linked to a heterologouspolynucleotide, and b) regenerating stably transformed plants from saidtransformed plant cell.
 22. A plant prepared according to the method ofclaim
 20. 23. Seed from the plant according to claim
 22. 24. A method ofproducing a transgenic plant, said method comprising: (a) introducinginto a plant cell: (i) an isolated polynucleotide comprising the nucleicacid according to claim 1 that is operably linked to a heterologouspolynucleotide, or (ii) the vector according to claim 7; and (b) growinga plant from said plant cell.
 25. The method of claim 24, wherein saidheterologous polynucleotide comprises a nucleic acid sequence encoding apolypeptide.
 26. The method of claim 24, wherein said heterologouspolynucleotide is operably linked to said regulatory region in theantisense orientation.
 27. The method of claim 24, wherein saidheterologous polynucleotide is transcribed into an interfering RNA.